Structured Review

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The NPC1 restores the cellular distribution of cholesterol and cav-1 in AnxA6-overexpressing cells. A) The COS-1 cells were cotransfected with Anx6–CFP (b) and NPC1–GFP (c), fixed, and analyzed by confocal microscopy. The merged image, which is representative for 90% of cells analyzed, is shown in panel a. Arrows in the enlarged sections (panels b and c) point at colocalization of AnxA6ndash;CFP and NPC1–GFP. Bar is 10 μm. B) The CHOanx6 cells were transfected with NPC1–GFP. Twenty four hours after transfection, cell lysates (WCL) were immunoprecipitated with or without (−) a polyclonal antibody against GFP (anti-GFP) as described (15) and analyzed by Western blotting for the presence of NPC1–GFP and AnxA6. C) The CHOanx6 cells were transfected with NPC1–GFP (b and d), fixed, permeabilized and immunolabeled with anti-caveolin (a) and stained with <t>filipin</t> (c). Cav-1 and cholesterol accumulation (arrows) in the Golgi and LE (panels a and c) is lost upon overexpression of NPC1 (arrowheads in panels a and c). Bar is 10 μm.
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Images

1) Product Images from "Annexin A6-Induced Alterations in Cholesterol Transport and Caveolin Export from the Golgi Complex"

Article Title: Annexin A6-Induced Alterations in Cholesterol Transport and Caveolin Export from the Golgi Complex

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/j.1600-0854.2007.00640.x

The NPC1 restores the cellular distribution of cholesterol and cav-1 in AnxA6-overexpressing cells. A) The COS-1 cells were cotransfected with Anx6–CFP (b) and NPC1–GFP (c), fixed, and analyzed by confocal microscopy. The merged image, which is representative for 90% of cells analyzed, is shown in panel a. Arrows in the enlarged sections (panels b and c) point at colocalization of AnxA6ndash;CFP and NPC1–GFP. Bar is 10 μm. B) The CHOanx6 cells were transfected with NPC1–GFP. Twenty four hours after transfection, cell lysates (WCL) were immunoprecipitated with or without (−) a polyclonal antibody against GFP (anti-GFP) as described (15) and analyzed by Western blotting for the presence of NPC1–GFP and AnxA6. C) The CHOanx6 cells were transfected with NPC1–GFP (b and d), fixed, permeabilized and immunolabeled with anti-caveolin (a) and stained with filipin (c). Cav-1 and cholesterol accumulation (arrows) in the Golgi and LE (panels a and c) is lost upon overexpression of NPC1 (arrowheads in panels a and c). Bar is 10 μm.
Figure Legend Snippet: The NPC1 restores the cellular distribution of cholesterol and cav-1 in AnxA6-overexpressing cells. A) The COS-1 cells were cotransfected with Anx6–CFP (b) and NPC1–GFP (c), fixed, and analyzed by confocal microscopy. The merged image, which is representative for 90% of cells analyzed, is shown in panel a. Arrows in the enlarged sections (panels b and c) point at colocalization of AnxA6ndash;CFP and NPC1–GFP. Bar is 10 μm. B) The CHOanx6 cells were transfected with NPC1–GFP. Twenty four hours after transfection, cell lysates (WCL) were immunoprecipitated with or without (−) a polyclonal antibody against GFP (anti-GFP) as described (15) and analyzed by Western blotting for the presence of NPC1–GFP and AnxA6. C) The CHOanx6 cells were transfected with NPC1–GFP (b and d), fixed, permeabilized and immunolabeled with anti-caveolin (a) and stained with filipin (c). Cav-1 and cholesterol accumulation (arrows) in the Golgi and LE (panels a and c) is lost upon overexpression of NPC1 (arrowheads in panels a and c). Bar is 10 μm.

Techniques Used: Confocal Microscopy, Transfection, Immunoprecipitation, Western Blot, Immunolabeling, Staining, Over Expression

Sequestration of cholesterol in the late endosomal compartment of AnxA6-expressing cells. A) The CHOwt and CHOanx6 cells grown on coverslips were fixed and stained with filipin (5 μg/mL) as indicated. Arrows and arrowheads point at the accumulation of cholesterol in perinuclear compartments and at the PM, respectively. B) The CHOwt cells transiently transfected with GFP–Anx6 (a and b), AnxA6-deficient A431 cells (A431wt) and A431 cells stably expressing AnxA6 (A431anx6) were fixed and stained with filipin (c and d). The arrows point at the accumulation of cholesterol in perinuclear vesicles of GFP–Anx6 expressing CHO and A431anx6 cells, compared with the non-transfected control (arrowheads). For the quantification in A (a and b) and B (a and b), see text for further details. The mean values ± SD of the diameter and fluorescence intensity of perinuclear filipin-positive structures from three independent experiments (150 cells in total) per cell line is given; ** and ***, p
Figure Legend Snippet: Sequestration of cholesterol in the late endosomal compartment of AnxA6-expressing cells. A) The CHOwt and CHOanx6 cells grown on coverslips were fixed and stained with filipin (5 μg/mL) as indicated. Arrows and arrowheads point at the accumulation of cholesterol in perinuclear compartments and at the PM, respectively. B) The CHOwt cells transiently transfected with GFP–Anx6 (a and b), AnxA6-deficient A431 cells (A431wt) and A431 cells stably expressing AnxA6 (A431anx6) were fixed and stained with filipin (c and d). The arrows point at the accumulation of cholesterol in perinuclear vesicles of GFP–Anx6 expressing CHO and A431anx6 cells, compared with the non-transfected control (arrowheads). For the quantification in A (a and b) and B (a and b), see text for further details. The mean values ± SD of the diameter and fluorescence intensity of perinuclear filipin-positive structures from three independent experiments (150 cells in total) per cell line is given; ** and ***, p

Techniques Used: Expressing, Staining, Transfection, Stable Transfection, Fluorescence

Sequestration of cholesterol in LE alters the cellular distribution of caveolin. A) The CHOwt and CHOanx6 cells were treated ± U18666A (2 μg/mL) for 24 h, fixed and stained with anti-caveolin, anti-LBPA and filipin (5 μg/mL) as indicated. A redistribution and increased cav-1 staining in the Golgi of U18666A-treated CHOwt can be observed (panels b and n). The merged images are shown (panels j–l). Bar is 10 μm. B and C) The CHOwt, CHOanx6 (B) and A431 (C) cells were treated ± U18666A as above, and Golgi-associated caveolin was immunoprecipitated and analyzed by Western blotting as described (40) . Actin in the cell lysates is shown. The amount of immunoprecipitated cav-1 from the Golgi was quantified and normalized to actin. The relative amount of cav-1 in the Golgi is given and is representative for three independent experiments. ***, p
Figure Legend Snippet: Sequestration of cholesterol in LE alters the cellular distribution of caveolin. A) The CHOwt and CHOanx6 cells were treated ± U18666A (2 μg/mL) for 24 h, fixed and stained with anti-caveolin, anti-LBPA and filipin (5 μg/mL) as indicated. A redistribution and increased cav-1 staining in the Golgi of U18666A-treated CHOwt can be observed (panels b and n). The merged images are shown (panels j–l). Bar is 10 μm. B and C) The CHOwt, CHOanx6 (B) and A431 (C) cells were treated ± U18666A as above, and Golgi-associated caveolin was immunoprecipitated and analyzed by Western blotting as described (40) . Actin in the cell lysates is shown. The amount of immunoprecipitated cav-1 from the Golgi was quantified and normalized to actin. The relative amount of cav-1 in the Golgi is given and is representative for three independent experiments. ***, p

Techniques Used: Staining, Immunoprecipitation, Western Blot

Expression of an N-terminal deletion mutant or knock down of AnxA6 restores the cellular distribution of cholesterol and cav-1. A) The CHOanx6 cells were transfected with Anx6 1–175 −myc (a and b), fixed, stained with filipin (5 μg/mL) (a) and immunolabeled with anti-myc (b) as indicated. Arrows point at vesicular, cholesterol-containing structures (a). Loss of strong perinuclear filipin staining was observed in 50% of transfected cells in five independent experiments and quantified. The mean ± SD of the relative filipin intensity in the perinuclear region is given. B) The CHOanx6 cells were transfected with Anx6 1–175 −myc (a and b) and empty vector (c and d). Twenty-four hours after transfection, cells were fixed, permeabilized and immunolabeled with anti-cav-1 (a and c) and anti-myc (b and d) as indicated. As shown in the schematic diagram, myc-tagged Anx6 1–175 contains the N-terminal signal peptide (blue) and two out of eight AnxA6-membrane-binding repeats (gray). Arrows point at the increased amount of cav-1 in the Golgi of non-transfected and vector-transfected CHOanx6 cells (a and c). Arrowheads point at reduced and unchanged cav-1 staining at the Golgi in Anx6 1–175 -transfected (panel a) and control cells (panel c), respectively. Reduced cav-1 staining in the Golgi region upon Anx6 1–175 expression was observed in 45–55% of transfected cells in five independent experiments. The fluorescence intensity of the cav-1 staining at the Golgi in Anx6 1–175 -transfected versus non-transfected (panel a) and myc-transfected cells (panel c) were quantified. Values represent the mean ± SD of 50 cells per cell line in each experiment ( n = 5). ***, p
Figure Legend Snippet: Expression of an N-terminal deletion mutant or knock down of AnxA6 restores the cellular distribution of cholesterol and cav-1. A) The CHOanx6 cells were transfected with Anx6 1–175 −myc (a and b), fixed, stained with filipin (5 μg/mL) (a) and immunolabeled with anti-myc (b) as indicated. Arrows point at vesicular, cholesterol-containing structures (a). Loss of strong perinuclear filipin staining was observed in 50% of transfected cells in five independent experiments and quantified. The mean ± SD of the relative filipin intensity in the perinuclear region is given. B) The CHOanx6 cells were transfected with Anx6 1–175 −myc (a and b) and empty vector (c and d). Twenty-four hours after transfection, cells were fixed, permeabilized and immunolabeled with anti-cav-1 (a and c) and anti-myc (b and d) as indicated. As shown in the schematic diagram, myc-tagged Anx6 1–175 contains the N-terminal signal peptide (blue) and two out of eight AnxA6-membrane-binding repeats (gray). Arrows point at the increased amount of cav-1 in the Golgi of non-transfected and vector-transfected CHOanx6 cells (a and c). Arrowheads point at reduced and unchanged cav-1 staining at the Golgi in Anx6 1–175 -transfected (panel a) and control cells (panel c), respectively. Reduced cav-1 staining in the Golgi region upon Anx6 1–175 expression was observed in 45–55% of transfected cells in five independent experiments. The fluorescence intensity of the cav-1 staining at the Golgi in Anx6 1–175 -transfected versus non-transfected (panel a) and myc-transfected cells (panel c) were quantified. Values represent the mean ± SD of 50 cells per cell line in each experiment ( n = 5). ***, p

Techniques Used: Expressing, Mutagenesis, Transfection, Staining, Immunolabeling, Plasmid Preparation, Binding Assay, Fluorescence

2) Product Images from "Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner"

Article Title: Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner

Journal: PLoS ONE

doi: 10.1371/journal.pone.0034795

Cholesterol depletion is detrimental to the HCMV entry into MDDCs. A) Cells were pre-incubated with filipin (7.66, 1.5, 0.3 µM), nystatin (21.2, 4.3, 0.85 µM) or methyl-β-cyclodextrin (MβCD; 5, 1, 0.2 mM) or with vehicle (DMSO, 1/100) and were processed as described in the legend for Figure 1D . For nystatin, n= 2 independent experiments with 2 different donors in total; for Filipin and MβCD n=4 independent experiments with 6 different donors in total. ns : not significant (p=0,0535).
Figure Legend Snippet: Cholesterol depletion is detrimental to the HCMV entry into MDDCs. A) Cells were pre-incubated with filipin (7.66, 1.5, 0.3 µM), nystatin (21.2, 4.3, 0.85 µM) or methyl-β-cyclodextrin (MβCD; 5, 1, 0.2 mM) or with vehicle (DMSO, 1/100) and were processed as described in the legend for Figure 1D . For nystatin, n= 2 independent experiments with 2 different donors in total; for Filipin and MβCD n=4 independent experiments with 6 different donors in total. ns : not significant (p=0,0535).

Techniques Used: Incubation

3) Product Images from "ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling"

Article Title: ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling

Journal: Scientific Reports

doi: 10.1038/srep26986

CIE but not CME of CTxB is impaired in Alix ko cells. ( a ) Representative images of 5 min CTxB-TRITC uptake by wt and Alix ko MEFs. The reduced endocytosis of CTxB-TRITC seen in Alix ko cells is further decreased by chlorpromazine (chlorpro) but not by filipin. Ø: no treatment. Hoechst stained nuclei appear in blue. ( b ) Quantification of the experiment shown in ( a ). Mean fluorescence values per cell were calculated using ImageJ (Number of cells in 3 independent experiments: wt Ø, n = 107; wt filipin, n = 90; wt chlorpro, n = 72; Alix ko Ø, n = 145; Alix ko filipin, n = 100; Alix ko chlorpro, n = 95; *P
Figure Legend Snippet: CIE but not CME of CTxB is impaired in Alix ko cells. ( a ) Representative images of 5 min CTxB-TRITC uptake by wt and Alix ko MEFs. The reduced endocytosis of CTxB-TRITC seen in Alix ko cells is further decreased by chlorpromazine (chlorpro) but not by filipin. Ø: no treatment. Hoechst stained nuclei appear in blue. ( b ) Quantification of the experiment shown in ( a ). Mean fluorescence values per cell were calculated using ImageJ (Number of cells in 3 independent experiments: wt Ø, n = 107; wt filipin, n = 90; wt chlorpro, n = 72; Alix ko Ø, n = 145; Alix ko filipin, n = 100; Alix ko chlorpro, n = 95; *P

Techniques Used: Staining, Fluorescence

4) Product Images from "Cholesterol Regulates Glucose-stimulated Insulin Secretion through Phosphatidylinositol 4,5-Bisphosphate *"

Article Title: Cholesterol Regulates Glucose-stimulated Insulin Secretion through Phosphatidylinositol 4,5-Bisphosphate *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.038034

F-actin content correlates with cellular cholesterol abundance. A , INS-1 β-cells overloaded with cholesterol were labeled with filipin (for cholesterol) and Alexa 546-phalloidin (for F-actin). The arrowheads show cells enriched with cholesterol,
Figure Legend Snippet: F-actin content correlates with cellular cholesterol abundance. A , INS-1 β-cells overloaded with cholesterol were labeled with filipin (for cholesterol) and Alexa 546-phalloidin (for F-actin). The arrowheads show cells enriched with cholesterol,

Techniques Used: Labeling

5) Product Images from "Annexin A6 and Late Endosomal Cholesterol Modulate Integrin Recycling and Cell Migration *"

Article Title: Annexin A6 and Late Endosomal Cholesterol Modulate Integrin Recycling and Cell Migration *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.683557

Accumulation of LE cholesterol reduces the association of Stx6 with the TGN. A and B , CHO-WT and CHO-A6 cells treated with 3 μg/ml U18666A ( A ) or 0–4.5 μg/ml U18666A for 16 h ( B ) were stained with anti-Stx6 and filipin. Insets
Figure Legend Snippet: Accumulation of LE cholesterol reduces the association of Stx6 with the TGN. A and B , CHO-WT and CHO-A6 cells treated with 3 μg/ml U18666A ( A ) or 0–4.5 μg/ml U18666A for 16 h ( B ) were stained with anti-Stx6 and filipin. Insets

Techniques Used: Staining

6) Product Images from "Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms ▿Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms ▿ †"

Article Title: Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms ▿Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms ▿ †

Journal: Infection and Immunity

doi: 10.1128/IAI.01835-06

Disruption of membrane microdomains inhibits uptake by epithelial CEACAMs. (A and B) 293T cells were transfected with the empty control vector (pcDNA) or cDNA encoding CEACAM3 or CEACAM6. Transfected cells were infected for 60 min with Opa CEA -expressing N. gonorrhoeae (MOI, 20) and applied in gentamicin assays. Prior to infection, cells were pretreated or not with the indicated amounts of nystatin (A) or filipin (B). The bars represent mean values ± standard deviations from three independent experiments done in triplicate. Values are expressed as percentages of the untreated control value. (C) 293T cells expressing the indicated CEACAMs were infected for 60 min with Opa CEA -expressing gonococci and analyzed in gentamicin assays in the presence or absence of 20 μg/ml nystatin. The bars represent mean values ± standard deviations from three independent experiments done in triplicate. Values are expressed as percentages of the untreated control value.
Figure Legend Snippet: Disruption of membrane microdomains inhibits uptake by epithelial CEACAMs. (A and B) 293T cells were transfected with the empty control vector (pcDNA) or cDNA encoding CEACAM3 or CEACAM6. Transfected cells were infected for 60 min with Opa CEA -expressing N. gonorrhoeae (MOI, 20) and applied in gentamicin assays. Prior to infection, cells were pretreated or not with the indicated amounts of nystatin (A) or filipin (B). The bars represent mean values ± standard deviations from three independent experiments done in triplicate. Values are expressed as percentages of the untreated control value. (C) 293T cells expressing the indicated CEACAMs were infected for 60 min with Opa CEA -expressing gonococci and analyzed in gentamicin assays in the presence or absence of 20 μg/ml nystatin. The bars represent mean values ± standard deviations from three independent experiments done in triplicate. Values are expressed as percentages of the untreated control value.

Techniques Used: Transfection, Plasmid Preparation, Infection, Expressing

7) Product Images from "Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides"

Article Title: Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

Journal: Molecular Therapy. Methods & Clinical Development

doi: 10.1038/mtm.2013.12

Entry mechanisms of adeno-associated virus type 2 (AAV2)-cell-permeable peptides (CPP) complexes. (a) The internalization of AAV2 or AAV2-CPP complexes in cells after 30 minutes incubation at 37 or 4 °C. (b) The effect of inhibitory drugs on viral transduction of cells infected by AAV2 or AAV2-CPP complexes. HEK293T cells were preincubated with chlorpromazine (CPZ, 30 nmol/l), filipin (15 nmol/l), or Amiloride (1 mmol/l) for 30 minutes at 37 °C. Treated and untreated cells were spin-infected with the AAV2 alone or complexes of AAV2 with peptides (final concentration: 200 μmol/l) for 90 minutes in the presence of drugs. Green flourescent protein (GFP) expression was analyzed 2 days after infection. All error bars represent the standard deviation of the mean from triplicate experiments. Asterisks indicate statistical significance compared to the no drug treatment group (* P
Figure Legend Snippet: Entry mechanisms of adeno-associated virus type 2 (AAV2)-cell-permeable peptides (CPP) complexes. (a) The internalization of AAV2 or AAV2-CPP complexes in cells after 30 minutes incubation at 37 or 4 °C. (b) The effect of inhibitory drugs on viral transduction of cells infected by AAV2 or AAV2-CPP complexes. HEK293T cells were preincubated with chlorpromazine (CPZ, 30 nmol/l), filipin (15 nmol/l), or Amiloride (1 mmol/l) for 30 minutes at 37 °C. Treated and untreated cells were spin-infected with the AAV2 alone or complexes of AAV2 with peptides (final concentration: 200 μmol/l) for 90 minutes in the presence of drugs. Green flourescent protein (GFP) expression was analyzed 2 days after infection. All error bars represent the standard deviation of the mean from triplicate experiments. Asterisks indicate statistical significance compared to the no drug treatment group (* P

Techniques Used: Conditioned Place Preference, Incubation, Transduction, Infection, Concentration Assay, Expressing, Standard Deviation

8) Product Images from "Natamycin Inhibits Vacuole Fusion at the Priming Phase via a Specific Interaction with Ergosterol ▿"

Article Title: Natamycin Inhibits Vacuole Fusion at the Priming Phase via a Specific Interaction with Ergosterol ▿

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01794-09

Effect of the polyene antibiotics natamycin, nystatin, and filipin on the fusion of isolated vacuoles. (A) Structures of natamycin (I), filipin (II), and nystatin (III). (B) Vacuoles isolated from yeasts with a wild-type sterol composition (3 μg
Figure Legend Snippet: Effect of the polyene antibiotics natamycin, nystatin, and filipin on the fusion of isolated vacuoles. (A) Structures of natamycin (I), filipin (II), and nystatin (III). (B) Vacuoles isolated from yeasts with a wild-type sterol composition (3 μg

Techniques Used: Isolation

Inhibitory staging assay of vacuole fusion with the polyene antibiotics filipin, nystatin, and natamycin. Vacuoles isolated from yeasts with a wild-type sterol composition (3 μg protein of KTY1 and KTY2 each) were incubated with different inhibitors
Figure Legend Snippet: Inhibitory staging assay of vacuole fusion with the polyene antibiotics filipin, nystatin, and natamycin. Vacuoles isolated from yeasts with a wild-type sterol composition (3 μg protein of KTY1 and KTY2 each) were incubated with different inhibitors

Techniques Used: Isolation, Incubation

9) Product Images from "Induction of Dendritic Cell Maturation and Activation by a Potential Adjuvant, 2-Hydroxypropyl-β-Cyclodextrin"

Article Title: Induction of Dendritic Cell Maturation and Activation by a Potential Adjuvant, 2-Hydroxypropyl-β-Cyclodextrin

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2016.00435

HP-β-CD triggers lipid raft formation in DCs . Human monocyte-derived DCs (2.5 × 10 5 cells/ml) were stimulated with HP-β-CD (1 mg/ml) in the presence or absence of filipin (30 μg/ml) or methanol as a vehicle control for 45 min. (A) Stimulated DCs were stained with Alexa Fluor ® 594-conjugated CTB, and the formation of lipid rafts on the cell surface was examined by confocal microscopy. (B) Fluorescence intensity of DCs was analyzed by ZEN software. DIC, differential interference contrast; AU, arbitrary unit. Images shown are representative of three similar results.
Figure Legend Snippet: HP-β-CD triggers lipid raft formation in DCs . Human monocyte-derived DCs (2.5 × 10 5 cells/ml) were stimulated with HP-β-CD (1 mg/ml) in the presence or absence of filipin (30 μg/ml) or methanol as a vehicle control for 45 min. (A) Stimulated DCs were stained with Alexa Fluor ® 594-conjugated CTB, and the formation of lipid rafts on the cell surface was examined by confocal microscopy. (B) Fluorescence intensity of DCs was analyzed by ZEN software. DIC, differential interference contrast; AU, arbitrary unit. Images shown are representative of three similar results.

Techniques Used: Derivative Assay, Staining, CtB Assay, Confocal Microscopy, Fluorescence, Software

Inhibition of lipid raft formation attenuates DC maturation, cytokine production, and autologous T cell activation induced by HP-β-CD . Human monocyte-derived DCs (2.5 × 10 5 cells/ml) were pretreated with filipin (10 and 30 μg/ml) or methanol as a vehicle control for 1 h, followed by stimulation with HP-β-CD (1 mg/ml) for another 24 h. (A–C) The expression of costimulatory molecules, MHC class II, and PD-L1/L2 was analyzed by flow cytometry. The numbers on the histograms indicate the MFIs of the cells. Scatter plots under the histogram show MFI of DCs ( n = 5). Statistical differences between compared groups were analyzed by paired Student’s t -test. N.S., not significant; * P
Figure Legend Snippet: Inhibition of lipid raft formation attenuates DC maturation, cytokine production, and autologous T cell activation induced by HP-β-CD . Human monocyte-derived DCs (2.5 × 10 5 cells/ml) were pretreated with filipin (10 and 30 μg/ml) or methanol as a vehicle control for 1 h, followed by stimulation with HP-β-CD (1 mg/ml) for another 24 h. (A–C) The expression of costimulatory molecules, MHC class II, and PD-L1/L2 was analyzed by flow cytometry. The numbers on the histograms indicate the MFIs of the cells. Scatter plots under the histogram show MFI of DCs ( n = 5). Statistical differences between compared groups were analyzed by paired Student’s t -test. N.S., not significant; * P

Techniques Used: Inhibition, Activation Assay, Derivative Assay, Expressing, Flow Cytometry, Cytometry

10) Product Images from "StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis [S]"

Article Title: StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M091967

StarD5 deletion lowers PM cholesterol in macrophages and ABCA1-dependent cholesterol efflux. A: Primary peritoneal macrophages from WT and StarD5 −/− mice were imaged following staining with filipin and BODIPY 493-503, as indicated, as well as by differential interference contrast for those stained with BODIPY 493-503. B: Total, free, esterified, and PM cholesterol were quantified as described in the Materials and Methods (n = 4). C: Accessible PM cholesterol was quantified as described in the Materials and Methods (n = 3, * P ≤ 0.05 for WT versus StarD5 −/− ). D: Primary peritoneal macrophages from WT and StarD5 −/− mice were used in cholesterol efflux assays as described in the Materials and Methods in the absence or presence of cAMP or cyclosporine A. Cholesterol efflux was calculated as the percentage of total cell 3 H-cholesterol content, which was about 5% of the total cholesterol (total effluxed 3 H-cholesterol + cell-associated 3 H-cholesterol) and represented as a percentage of the effluxed cholesterol in WT macrophages in the presence of cAMP. Primary peritoneal macrophages from StarD5 −/− mice infected with Ad-StarD5 (MOI of 200) were also used. The values represent the average ± SD (n = 4, * P
Figure Legend Snippet: StarD5 deletion lowers PM cholesterol in macrophages and ABCA1-dependent cholesterol efflux. A: Primary peritoneal macrophages from WT and StarD5 −/− mice were imaged following staining with filipin and BODIPY 493-503, as indicated, as well as by differential interference contrast for those stained with BODIPY 493-503. B: Total, free, esterified, and PM cholesterol were quantified as described in the Materials and Methods (n = 4). C: Accessible PM cholesterol was quantified as described in the Materials and Methods (n = 3, * P ≤ 0.05 for WT versus StarD5 −/− ). D: Primary peritoneal macrophages from WT and StarD5 −/− mice were used in cholesterol efflux assays as described in the Materials and Methods in the absence or presence of cAMP or cyclosporine A. Cholesterol efflux was calculated as the percentage of total cell 3 H-cholesterol content, which was about 5% of the total cholesterol (total effluxed 3 H-cholesterol + cell-associated 3 H-cholesterol) and represented as a percentage of the effluxed cholesterol in WT macrophages in the presence of cAMP. Primary peritoneal macrophages from StarD5 −/− mice infected with Ad-StarD5 (MOI of 200) were also used. The values represent the average ± SD (n = 4, * P

Techniques Used: Mouse Assay, Staining, Infection

11) Product Images from "Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection"

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection

Journal: MicrobiologyOpen

doi: 10.1002/mbo3.469

Fluorophore‐labeled cholesterol does not accumulate around the parasite. (a) Macrophages were preincubated with NBD ‐ or TopFluor‐cholesterol for 16 hr. Afterward, macrophages were infected with Leishmania mexicana amastigotes ( MOI of 5) for 2 hr. Seventy‐two hours after infection, cells were fixed and subsequently stained with DAPI (nuclei, excited at 405 nm). Both NBD and TopFluor groups were excited at 488 nm. (b) Distribution of filipin‐stainable and fluorophore‐labeled cholesterol in the early phases of infection with L . mexicana . Mouse bone marrow‐derived macrophages were preincubated with NBD ‐cholesterol for 16 hr and then infected with L . mexicana amastigotes ( MOI of 5) for 2 hr. An hour after infection, cells were fixed and subsequently stained with filipin (excited at 405 nm). For a better identification, filipin‐stained cholesterol in the presence of NBD ‐cholesterol is visualized in purple. Arrows indicate Leishmania parasites and/or filipin around the parasites. All images are confocal and were acquired using a Zeiss LSM 780 microscope, 63× oil‐immersion objective, and processed using identical conditions. (c) Relative fluorescence corresponding to NBD ‐cholesterol incorporated by all the parasites (left panel) or a single parasite (right panel) in an infected macrophage is represented as a function of the number of parasites internalized by the macrophages over a period of 48 hr postinfection. The exponential curve regressions were calculated using GraphPad Prism
Figure Legend Snippet: Fluorophore‐labeled cholesterol does not accumulate around the parasite. (a) Macrophages were preincubated with NBD ‐ or TopFluor‐cholesterol for 16 hr. Afterward, macrophages were infected with Leishmania mexicana amastigotes ( MOI of 5) for 2 hr. Seventy‐two hours after infection, cells were fixed and subsequently stained with DAPI (nuclei, excited at 405 nm). Both NBD and TopFluor groups were excited at 488 nm. (b) Distribution of filipin‐stainable and fluorophore‐labeled cholesterol in the early phases of infection with L . mexicana . Mouse bone marrow‐derived macrophages were preincubated with NBD ‐cholesterol for 16 hr and then infected with L . mexicana amastigotes ( MOI of 5) for 2 hr. An hour after infection, cells were fixed and subsequently stained with filipin (excited at 405 nm). For a better identification, filipin‐stained cholesterol in the presence of NBD ‐cholesterol is visualized in purple. Arrows indicate Leishmania parasites and/or filipin around the parasites. All images are confocal and were acquired using a Zeiss LSM 780 microscope, 63× oil‐immersion objective, and processed using identical conditions. (c) Relative fluorescence corresponding to NBD ‐cholesterol incorporated by all the parasites (left panel) or a single parasite (right panel) in an infected macrophage is represented as a function of the number of parasites internalized by the macrophages over a period of 48 hr postinfection. The exponential curve regressions were calculated using GraphPad Prism

Techniques Used: Labeling, Infection, Staining, Derivative Assay, Microscopy, Fluorescence

Filipin accumulation in macrophages upon Leishmania mexicana amastigotes infection. Macrophages were infected with L. mexicana amastigotes expressing DsRed ( MOI of 5) for 2 hr. Distribution of filipin was analyzed over a time course of 96 hr, with time points every 24 hr. Cells were fixed and subsequently stained with filipin (white, excited at 405 nm) and SYTO ® 13 (nuclei, green, excited at 488 nm). Arrows indicate the presence of Leishmania parasites (red) and/or filipin around the parasites. One representative of three independent experiments is shown. All images are confocal and were acquired using a Zeiss LSM 780 microscope, 63× oil‐immersion objective, and processed using identical conditions
Figure Legend Snippet: Filipin accumulation in macrophages upon Leishmania mexicana amastigotes infection. Macrophages were infected with L. mexicana amastigotes expressing DsRed ( MOI of 5) for 2 hr. Distribution of filipin was analyzed over a time course of 96 hr, with time points every 24 hr. Cells were fixed and subsequently stained with filipin (white, excited at 405 nm) and SYTO ® 13 (nuclei, green, excited at 488 nm). Arrows indicate the presence of Leishmania parasites (red) and/or filipin around the parasites. One representative of three independent experiments is shown. All images are confocal and were acquired using a Zeiss LSM 780 microscope, 63× oil‐immersion objective, and processed using identical conditions

Techniques Used: Infection, Expressing, Staining, Microscopy

Distribution and quantification of the filipin signal around Leishmania mexicana amastigotes and latex beads. Macrophages were simultaneously infected with L . mexicana amastigotes ( MOI of 5) and incubated with latex beads (five per cell) for 2 hr. Distribution of filipin was analyzed 72 hr after infection. Cells were fixed and subsequently stained with filipin (white, excited at 405 nm) and SYTO ® 13 (nuclei, green, excited at 488 nm). Latex beads are visualized using the transmission channel. The image represents a confocal section acquired using a Zeiss LSM 780 microscope, 63× oil‐immersion objective. (left panel) Arrows indicate the presence Leishmania parasites (red, excited at 543 nm) and/or filipin‐stainable free cholesterol around the parasites. Arrowheads indicate selected latex beads incorporated by macrophages. One representative of three independent experiments is shown. (right panel top) Red arrows and charts outlined in red refer to representative linescans (measured with ZEN software) of L . mexicana parasites phagocytosed by macrophage. Arrows and charts outlined in blue refer to representative linescans of latex beads incorporated by macrophages. White arrows indicate signal intensities for filipin (white line) around latex beads (yellow line) and parasites (red line) that were used to quantify intensity differences in filipin staining. (right panel bottom) For quantification of filipin signal around internalized L . mexicana amastigotes and latex beads, maximal signal intensity (indicated as mean peak value in arbitrary units ( AU ) ± SD , n = 36 along section paths) was determined using ZEN (blue edition) software. Background values were subtracted from the absolute values. Differences between parasites and latex beads were highly significant p
Figure Legend Snippet: Distribution and quantification of the filipin signal around Leishmania mexicana amastigotes and latex beads. Macrophages were simultaneously infected with L . mexicana amastigotes ( MOI of 5) and incubated with latex beads (five per cell) for 2 hr. Distribution of filipin was analyzed 72 hr after infection. Cells were fixed and subsequently stained with filipin (white, excited at 405 nm) and SYTO ® 13 (nuclei, green, excited at 488 nm). Latex beads are visualized using the transmission channel. The image represents a confocal section acquired using a Zeiss LSM 780 microscope, 63× oil‐immersion objective. (left panel) Arrows indicate the presence Leishmania parasites (red, excited at 543 nm) and/or filipin‐stainable free cholesterol around the parasites. Arrowheads indicate selected latex beads incorporated by macrophages. One representative of three independent experiments is shown. (right panel top) Red arrows and charts outlined in red refer to representative linescans (measured with ZEN software) of L . mexicana parasites phagocytosed by macrophage. Arrows and charts outlined in blue refer to representative linescans of latex beads incorporated by macrophages. White arrows indicate signal intensities for filipin (white line) around latex beads (yellow line) and parasites (red line) that were used to quantify intensity differences in filipin staining. (right panel bottom) For quantification of filipin signal around internalized L . mexicana amastigotes and latex beads, maximal signal intensity (indicated as mean peak value in arbitrary units ( AU ) ± SD , n = 36 along section paths) was determined using ZEN (blue edition) software. Background values were subtracted from the absolute values. Differences between parasites and latex beads were highly significant p

Techniques Used: Infection, Incubation, Staining, Transmission Assay, Microscopy, Software

Amount of sequestered filipin per parasite is a function of the number of parasites per cell. Relative filipin signal of (a) confocal 2D sections and (b) 3D confocal microscope image stack processed in a 2D projection by maximum intensity projection is represented as a function of the number of parasites internalized by the macrophages over a period of 96 hr postinfection. The exponential curve regressions were calculated using GraphPad Prism
Figure Legend Snippet: Amount of sequestered filipin per parasite is a function of the number of parasites per cell. Relative filipin signal of (a) confocal 2D sections and (b) 3D confocal microscope image stack processed in a 2D projection by maximum intensity projection is represented as a function of the number of parasites internalized by the macrophages over a period of 96 hr postinfection. The exponential curve regressions were calculated using GraphPad Prism

Techniques Used: Microscopy

12) Product Images from "Transcriptional profile of Paracoccidioides spp. in response to itraconazole"

Article Title: Transcriptional profile of Paracoccidioides spp. in response to itraconazole

Journal: BMC Genomics

doi: 10.1186/1471-2164-15-254

Sterol distribution in Paracoccidioides spp.. Yeast cells were fixed, stained with filipin and observed by fluorescence microscopy. Staining in the control Pb 01 (A) and Pb 18 (C) cells was diffuse with homogeneous labeling. Pb 01 (B) and Pb 18 (D) cells treated with itraconazole displayed heterogeneous fluorescence.
Figure Legend Snippet: Sterol distribution in Paracoccidioides spp.. Yeast cells were fixed, stained with filipin and observed by fluorescence microscopy. Staining in the control Pb 01 (A) and Pb 18 (C) cells was diffuse with homogeneous labeling. Pb 01 (B) and Pb 18 (D) cells treated with itraconazole displayed heterogeneous fluorescence.

Techniques Used: Staining, Fluorescence, Microscopy, Labeling

13) Product Images from "Membrane cholesterol mediates the cellular effects of monolayer graphene substrates"

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates

Journal: Nature Communications

doi: 10.1038/s41467-018-03185-0

Graphene enhances P2Y receptor-mediated Ca 2+ responses. a Sample images of Filipin staining of 3T3 cells on graphene with (purple) or without (red) MβCD treatment and on glass with (green) or without (black) TFC loading. Same color coding hereafter. Scale bar, 50 μm. b Average intensities of Filipin staining ( n graphene = 1536 cells, n graphene + MBCD = 2286 cells, n glass = 1317 cells, n glass + TFC = 1487 cells, N = 3 batches for every group; for graphene vs. graphene+MβCD, graphene vs. glass, and glass vs. glass+TFC, *** p
Figure Legend Snippet: Graphene enhances P2Y receptor-mediated Ca 2+ responses. a Sample images of Filipin staining of 3T3 cells on graphene with (purple) or without (red) MβCD treatment and on glass with (green) or without (black) TFC loading. Same color coding hereafter. Scale bar, 50 μm. b Average intensities of Filipin staining ( n graphene = 1536 cells, n graphene + MBCD = 2286 cells, n glass = 1317 cells, n glass + TFC = 1487 cells, N = 3 batches for every group; for graphene vs. graphene+MβCD, graphene vs. glass, and glass vs. glass+TFC, *** p

Techniques Used: Staining

Cholesterol mediates graphene-induced presynaptic changes. a Sample Filipin staining images of neurites on graphene with (purple) or without (red) MβCD treatment and on glass with (green) or without (black) TFC loading. Scale bar, 50 μm. Same color coding hereafter. b Average Filipin staining intensities in neurites ( n graphene = 107 neurites, n graphene+MβCD = 151 neurites, n glass = 188 neurites, n glass+TFC = 152 neurites, N = 3 batches for every group; for graphene vs. graphene+MβCD, graphene vs. glass, and glass vs. glass+TFC, *** p
Figure Legend Snippet: Cholesterol mediates graphene-induced presynaptic changes. a Sample Filipin staining images of neurites on graphene with (purple) or without (red) MβCD treatment and on glass with (green) or without (black) TFC loading. Scale bar, 50 μm. Same color coding hereafter. b Average Filipin staining intensities in neurites ( n graphene = 107 neurites, n graphene+MβCD = 151 neurites, n glass = 188 neurites, n glass+TFC = 152 neurites, N = 3 batches for every group; for graphene vs. graphene+MβCD, graphene vs. glass, and glass vs. glass+TFC, *** p

Techniques Used: Staining

Graphene increases cell membrane cholesterol. a Sample images of Filipin staining. Scale bar, 100 μm. b Filipin fluorescence intensity in neuronal neurites ( n glass = 118 and n graphene = 112 neurites, N = 3 batches, *** p
Figure Legend Snippet: Graphene increases cell membrane cholesterol. a Sample images of Filipin staining. Scale bar, 100 μm. b Filipin fluorescence intensity in neuronal neurites ( n glass = 118 and n graphene = 112 neurites, N = 3 batches, *** p

Techniques Used: Staining, Fluorescence

14) Product Images from "Progesterone Impairs Human Ether-a-go-go-related Gene (HERG) Trafficking by Disruption of Intracellular Cholesterol Homeostasis *"

Article Title: Progesterone Impairs Human Ether-a-go-go-related Gene (HERG) Trafficking by Disruption of Intracellular Cholesterol Homeostasis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.198853

Effect of P4 on the distribution and level of cholesterol in HEK293 cells. A and B , filipin staining of intracellular free cholesterol is shown. A , P4 induced accumulation of cholesterol in a dose-dependent manner. B , sterol-binding agent HPCD (10 mg/ml)
Figure Legend Snippet: Effect of P4 on the distribution and level of cholesterol in HEK293 cells. A and B , filipin staining of intracellular free cholesterol is shown. A , P4 induced accumulation of cholesterol in a dose-dependent manner. B , sterol-binding agent HPCD (10 mg/ml)

Techniques Used: Staining, Binding Assay

15) Product Images from "Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol"

Article Title: Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1706070114

Ezetimibe is protective in a zebrafish model of S . Typhi infection. ( A ) Fish infected with S . Typhi prgH had increased survival compared with fish infected with WT S . Typhi ( P = 0.01). The survival curve was carried out for 5 d; fish were checked once each day. ( B ) Zebrafish were scored 24 h post S . Typhi infection as cleared (no bacteria), localized (bacteria only in the swim bladder), disseminated (bacteria found outside the swim bladder), or dead (fish dead due to bacterial burden). The swim bladders are denoted by red circles; bacteria are denoted by the red arrows. ( C ) Fish infected with the S . Typhi prgH mutant had increased clearance of bacteria at 24 h ( P = 0.03). ( D ) Ezetimibe had no effect on S . Typhi bacterial growth. Bacteria were diluted from an overnight stock and grown with DMSO or 10 µM ezetimibe. The OD 600 was taken every 30 min for 3.5 h. Data points are the mean from two separate experiments. ( E ) Ezetimibe decreased filipin staining in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe reduced filipin (0.05 mg/mL) staining ( P = 0.003) in zebrafish; n = 20 fish from two separate experiments; P value from an unpaired t test. ( F ) Fish pretreated with ezetimibe had increased survival from S . Typhi infection compared with DMSO-pretreated controls ( P = 0.03). ( G ) Ezetimibe treatment increased bacterial clearance in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe increased the percentage of fish that cleared the bacteria 24 h postinfection from 8 to 30% ( P = 0.009). Infection data for each survival curve and clearance comparisons are from three independent experiments with a minimum of n = 60 fish. P values from survival curves are from the Mantel–Cox test; P values for other comparisons are from unpaired t tests.
Figure Legend Snippet: Ezetimibe is protective in a zebrafish model of S . Typhi infection. ( A ) Fish infected with S . Typhi prgH had increased survival compared with fish infected with WT S . Typhi ( P = 0.01). The survival curve was carried out for 5 d; fish were checked once each day. ( B ) Zebrafish were scored 24 h post S . Typhi infection as cleared (no bacteria), localized (bacteria only in the swim bladder), disseminated (bacteria found outside the swim bladder), or dead (fish dead due to bacterial burden). The swim bladders are denoted by red circles; bacteria are denoted by the red arrows. ( C ) Fish infected with the S . Typhi prgH mutant had increased clearance of bacteria at 24 h ( P = 0.03). ( D ) Ezetimibe had no effect on S . Typhi bacterial growth. Bacteria were diluted from an overnight stock and grown with DMSO or 10 µM ezetimibe. The OD 600 was taken every 30 min for 3.5 h. Data points are the mean from two separate experiments. ( E ) Ezetimibe decreased filipin staining in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe reduced filipin (0.05 mg/mL) staining ( P = 0.003) in zebrafish; n = 20 fish from two separate experiments; P value from an unpaired t test. ( F ) Fish pretreated with ezetimibe had increased survival from S . Typhi infection compared with DMSO-pretreated controls ( P = 0.03). ( G ) Ezetimibe treatment increased bacterial clearance in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe increased the percentage of fish that cleared the bacteria 24 h postinfection from 8 to 30% ( P = 0.009). Infection data for each survival curve and clearance comparisons are from three independent experiments with a minimum of n = 60 fish. P values from survival curves are from the Mantel–Cox test; P values for other comparisons are from unpaired t tests.

Techniques Used: Infection, Fluorescence In Situ Hybridization, Mutagenesis, Staining

16) Product Images from "Histone deacetylase inhibitors modulate KATP subunit transcription in HL-1 cardiomyocytes through effects on cholesterol homeostasis"

Article Title: Histone deacetylase inhibitors modulate KATP subunit transcription in HL-1 cardiomyocytes through effects on cholesterol homeostasis

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2015.00168

TSA depletes cholesterol in HL-1 cells, but not MIN6 cells. (A) Representative images of HL-1 or MIN6 cells labeled with filipin (Cholesterol, blue) and picoGreen (Nuclei, green). (B) Treatment with TSA (30 ng/mL) markedly reduces filipin staining in HL-1 cells but has little effect on filipin staining in MIN6 cells. Scale bar = 25 μm.
Figure Legend Snippet: TSA depletes cholesterol in HL-1 cells, but not MIN6 cells. (A) Representative images of HL-1 or MIN6 cells labeled with filipin (Cholesterol, blue) and picoGreen (Nuclei, green). (B) Treatment with TSA (30 ng/mL) markedly reduces filipin staining in HL-1 cells but has little effect on filipin staining in MIN6 cells. Scale bar = 25 μm.

Techniques Used: Labeling, Staining

17) Product Images from "Host Complement Regulatory Protein CD59 Is Transported to the Chlamydial Inclusion by a Golgi Apparatus-Independent Pathway ▿"

Article Title: Host Complement Regulatory Protein CD59 Is Transported to the Chlamydial Inclusion by a Golgi Apparatus-Independent Pathway ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.01062-08

CD59 localizes to the membrane of the inclusion. (A) HeLa cells infected with C. trachomatis were stained with antibodies to CD59 and IncA (upper panels), with additional images obtained by confocal microscopy (lower panels). In the merged images, CD59 is shown in green, IncA is shown in red, and chlamydial and host cell DNA, stained by the Hoechst dye 33342, is shown in blue. Bar, 10 μm. (B) Chlamydia -infected HeLa cells stained with antibodies to CD59 and the fluorescent cholesterol-binding compound filipin. In the merged image, CD59 is shown in green, filipin is shown in blue, and chlamydiae stained with anti- Chlamydia antibodies are shown in red. Inclusions in which CD59 and filipin costained are marked by arrows. Bar, 10 μm.
Figure Legend Snippet: CD59 localizes to the membrane of the inclusion. (A) HeLa cells infected with C. trachomatis were stained with antibodies to CD59 and IncA (upper panels), with additional images obtained by confocal microscopy (lower panels). In the merged images, CD59 is shown in green, IncA is shown in red, and chlamydial and host cell DNA, stained by the Hoechst dye 33342, is shown in blue. Bar, 10 μm. (B) Chlamydia -infected HeLa cells stained with antibodies to CD59 and the fluorescent cholesterol-binding compound filipin. In the merged image, CD59 is shown in green, filipin is shown in blue, and chlamydiae stained with anti- Chlamydia antibodies are shown in red. Inclusions in which CD59 and filipin costained are marked by arrows. Bar, 10 μm.

Techniques Used: Infection, Staining, Confocal Microscopy, Binding Assay

18) Product Images from "Nogo-B Receptor stabilizes Niemann-Pick Type C2 protein and regulates intracellular cholesterol trafficking"

Article Title: Nogo-B Receptor stabilizes Niemann-Pick Type C2 protein and regulates intracellular cholesterol trafficking

Journal: Cell metabolism

doi: 10.1016/j.cmet.2009.07.003

Loss of NgBR induces free cholesterol accumulation in cells (A) EA.hy926 cells or (B) NgBR +/− fibroblasts (MEFs) were incubated with non-silencing RNA (Ctrl RNAi) or small interfering RNA directed against NgBR (NgBR RNAi) for 48 hours. U18666A (1µM, 8hours) was used as a positive control for inhibition of cholesterol trafficking. Cells were fixed and stained for free cholesterol with filipin as described in Experimental Procedures (scale bar = 20µm). (C) EA.hy926 cells were infected with an adenoviral construct expressing GFP or with adenovirus encoding NgBR (Ad-NgBR) for 24 hours, followed by incubation with Ctrl RNAi or NgBR RNAi for 48 hours. Cells were then fixed and stained for free cholesterol as in (A–B). (D) Conditioned medium from CHO cells transfected with NPC2-myc was concentrated and added to EA.hy926 cells incubated with Ctrl RNAi or NgBR RNAi for 24 hours. (E) EA.hy926 cells treated with Ctrl RNAi or NgBR RNAi were incubated with 2.5µM 25-hydroxycholesterol (25-HC) for 18 hours followed by fixation and staining with filipin. (*p
Figure Legend Snippet: Loss of NgBR induces free cholesterol accumulation in cells (A) EA.hy926 cells or (B) NgBR +/− fibroblasts (MEFs) were incubated with non-silencing RNA (Ctrl RNAi) or small interfering RNA directed against NgBR (NgBR RNAi) for 48 hours. U18666A (1µM, 8hours) was used as a positive control for inhibition of cholesterol trafficking. Cells were fixed and stained for free cholesterol with filipin as described in Experimental Procedures (scale bar = 20µm). (C) EA.hy926 cells were infected with an adenoviral construct expressing GFP or with adenovirus encoding NgBR (Ad-NgBR) for 24 hours, followed by incubation with Ctrl RNAi or NgBR RNAi for 48 hours. Cells were then fixed and stained for free cholesterol as in (A–B). (D) Conditioned medium from CHO cells transfected with NPC2-myc was concentrated and added to EA.hy926 cells incubated with Ctrl RNAi or NgBR RNAi for 24 hours. (E) EA.hy926 cells treated with Ctrl RNAi or NgBR RNAi were incubated with 2.5µM 25-hydroxycholesterol (25-HC) for 18 hours followed by fixation and staining with filipin. (*p

Techniques Used: Incubation, Small Interfering RNA, Positive Control, Inhibition, Staining, Infection, Construct, Expressing, Transfection

19) Product Images from "Characterization of a Nonclathrin Endocytic Pathway: Membrane Cargo and Lipid Requirements D⃞"

Article Title: Characterization of a Nonclathrin Endocytic Pathway: Membrane Cargo and Lipid Requirements D⃞

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E04-02-0151

CD59 and cholesterol reach Rab5 compartment via Arf6-endosomes. HeLa cells were transfected with GFP-Rab5-Q79L alone (A and C) or with GFP-Rab5-Q79L and Arf6-Q67L (B and D). In A and B, cells were allowed to internalize phycoerythrin anti-CD59 antibody at 37°C for 1 h. In C and D, cells were fixed and stained with filipin. Bar, 10 μm.
Figure Legend Snippet: CD59 and cholesterol reach Rab5 compartment via Arf6-endosomes. HeLa cells were transfected with GFP-Rab5-Q79L alone (A and C) or with GFP-Rab5-Q79L and Arf6-Q67L (B and D). In A and B, cells were allowed to internalize phycoerythrin anti-CD59 antibody at 37°C for 1 h. In C and D, cells were fixed and stained with filipin. Bar, 10 μm.

Techniques Used: Transfection, Staining

Effect of endocytosis inhibitors on CD59 and MHCI internalization. (A and B) HeLa cells were transfected with FLAG-c-AP180 and internalization of anti-CD59 and anti-MHCI (A) or of anti-MHCI and Tfn (B) was assessed. (C and D) Untransfected HeLa cells were preincubated for 20 min at 37°C with DMEM containing 0.5% BSA in the absence (C) or presence (D) of filipin (12 μg/ml). Then, Cy5-anti MHCI, R-phycoerythrin-anti-CD59, and Alexa 488-Tfn (5 μg/ml) were added for a 20-min uptake. The cells were fixed and processed for immunofluorescence. All images were taken with identical acquisition parameters and the amount of internalized antibody for either c-AP180 (E) or filipin treatment (F) was measured and expressed as a percentage of that internalized in control cells (see MATERIALS AND METHODS). Bar, 10 μm (A and B) and 20 μm (C and D).
Figure Legend Snippet: Effect of endocytosis inhibitors on CD59 and MHCI internalization. (A and B) HeLa cells were transfected with FLAG-c-AP180 and internalization of anti-CD59 and anti-MHCI (A) or of anti-MHCI and Tfn (B) was assessed. (C and D) Untransfected HeLa cells were preincubated for 20 min at 37°C with DMEM containing 0.5% BSA in the absence (C) or presence (D) of filipin (12 μg/ml). Then, Cy5-anti MHCI, R-phycoerythrin-anti-CD59, and Alexa 488-Tfn (5 μg/ml) were added for a 20-min uptake. The cells were fixed and processed for immunofluorescence. All images were taken with identical acquisition parameters and the amount of internalized antibody for either c-AP180 (E) or filipin treatment (F) was measured and expressed as a percentage of that internalized in control cells (see MATERIALS AND METHODS). Bar, 10 μm (A and B) and 20 μm (C and D).

Techniques Used: Transfection, Immunofluorescence

Tac-GPI, accumulated in Arf6-Q67L–induced vacuoles, is within detergent-resistant membranes. Cells coexpressing Arf6-Q67L and Tac-GPI were allowed to internalize anti-Tac antibody at 37°C for 1 h, cooled on ice, acid stripped, and incubated 3 min with ice-cold PBS (A) or with PBS and 1% TX-100 (B). After fixation, Arf6 was stained with polyclonal antibody. Tac-GPI was visualized with 488 Alexa goat anti-mouse, followed by staining of the free cholesterol with filipin. Bar, 10 μm.
Figure Legend Snippet: Tac-GPI, accumulated in Arf6-Q67L–induced vacuoles, is within detergent-resistant membranes. Cells coexpressing Arf6-Q67L and Tac-GPI were allowed to internalize anti-Tac antibody at 37°C for 1 h, cooled on ice, acid stripped, and incubated 3 min with ice-cold PBS (A) or with PBS and 1% TX-100 (B). After fixation, Arf6 was stained with polyclonal antibody. Tac-GPI was visualized with 488 Alexa goat anti-mouse, followed by staining of the free cholesterol with filipin. Bar, 10 μm.

Techniques Used: Incubation, Staining

Accelerated endocytosis of GPI-AP and clathrin-independent cargo into Arf6-Q67L expressing cells. (A) Cells transfected with Tac-GPI, Tac or Tac-LL alone (black bars) or in combination with Arf6-wt (white bars), Arf6-Q67L (red bars), or Arf6-T27N (blue bars) were incubated with biotinylated anti-Tac for 20 min at 37°C. Cells expressing Tac-GPI, Tac, or Tac-LL also were treated with filipin before Tac-antibody internalization (green bars). The surface-bound antibody was then removed by a low pH solution rinse, and the sample was fixed and processed for CELISA (see MATERIALS AND METHODS). (B and C) Internalization of Tac-GPI and Tac (circles and triangles in B), and Tac-LL (squares in C) was measured in a continuous uptake by using CELISA. Cells expressing the cargo proteins alone (black symbols) or with Arf6-Q67L (red symbols) were incubated at 37°C with biotinylated anti-Tac for the indicated time and then processed as described above. The amount of internalized Tac antibody is expressed as a percentage of total cell-associated anti-Tac. This experiment was performed three times with similar results. Data are means of triplicates with SD.
Figure Legend Snippet: Accelerated endocytosis of GPI-AP and clathrin-independent cargo into Arf6-Q67L expressing cells. (A) Cells transfected with Tac-GPI, Tac or Tac-LL alone (black bars) or in combination with Arf6-wt (white bars), Arf6-Q67L (red bars), or Arf6-T27N (blue bars) were incubated with biotinylated anti-Tac for 20 min at 37°C. Cells expressing Tac-GPI, Tac, or Tac-LL also were treated with filipin before Tac-antibody internalization (green bars). The surface-bound antibody was then removed by a low pH solution rinse, and the sample was fixed and processed for CELISA (see MATERIALS AND METHODS). (B and C) Internalization of Tac-GPI and Tac (circles and triangles in B), and Tac-LL (squares in C) was measured in a continuous uptake by using CELISA. Cells expressing the cargo proteins alone (black symbols) or with Arf6-Q67L (red symbols) were incubated at 37°C with biotinylated anti-Tac for the indicated time and then processed as described above. The amount of internalized Tac antibody is expressed as a percentage of total cell-associated anti-Tac. This experiment was performed three times with similar results. Data are means of triplicates with SD.

Techniques Used: Expressing, Transfection, Incubation

CD59 and cholesterol accumulate in Arf6-Q67L-associated vacuolar endosomes, devoid of caveolin-1. (A) Cells expressing Arf6-Q67L were incubated with anti-CD59 at 37°C for 1 h, and surface antibody was removed and then processed for immunofluorescence. After fixation, free cholesterol was stained with filipin as described in MATERIALS AND METHODS. Note the redistribution and intensity of filipin in the transfected cells (encircled). (B) Cells expressing Arf6-Q67L were labeled with antibody to caveolin-1. Bar, 10 μm.
Figure Legend Snippet: CD59 and cholesterol accumulate in Arf6-Q67L-associated vacuolar endosomes, devoid of caveolin-1. (A) Cells expressing Arf6-Q67L were incubated with anti-CD59 at 37°C for 1 h, and surface antibody was removed and then processed for immunofluorescence. After fixation, free cholesterol was stained with filipin as described in MATERIALS AND METHODS. Note the redistribution and intensity of filipin in the transfected cells (encircled). (B) Cells expressing Arf6-Q67L were labeled with antibody to caveolin-1. Bar, 10 μm.

Techniques Used: Expressing, Incubation, Immunofluorescence, Staining, Transfection, Labeling

20) Product Images from "High-density lipoprotein contribute to G0-G1/S transition in Swiss NIH/3T3 fibroblasts"

Article Title: High-density lipoprotein contribute to G0-G1/S transition in Swiss NIH/3T3 fibroblasts

Journal: Scientific Reports

doi: 10.1038/srep17812

Scratch wound assay. The figure reports DiI-HDL uptake, DiI-LDL binding, SR-BI immunocytofluorescence detection, the merged colour images obtained by Nile Red/filipin double staining, that show polar lipids and cytoplasmic membranes in red, LDs in green/yellow and plasma membrane cholesterol (filipin) in blue. The figure shows also BrdU (red) incorporated in DNA (Hoechst, blue). All detections were performed in distant (quiescent) and facing (proliferating) 3T3 Swiss mouse fibroblasts after 24 h from scratch-wound. Each experiment was carried out at least twice and the relative graphs and histograms show the mean values ± SE for each single fluorescence emission signal evaluated in at least 200 cells for each group. Bar is 30 μm. *p
Figure Legend Snippet: Scratch wound assay. The figure reports DiI-HDL uptake, DiI-LDL binding, SR-BI immunocytofluorescence detection, the merged colour images obtained by Nile Red/filipin double staining, that show polar lipids and cytoplasmic membranes in red, LDs in green/yellow and plasma membrane cholesterol (filipin) in blue. The figure shows also BrdU (red) incorporated in DNA (Hoechst, blue). All detections were performed in distant (quiescent) and facing (proliferating) 3T3 Swiss mouse fibroblasts after 24 h from scratch-wound. Each experiment was carried out at least twice and the relative graphs and histograms show the mean values ± SE for each single fluorescence emission signal evaluated in at least 200 cells for each group. Bar is 30 μm. *p

Techniques Used: Scratch Wound Assay Assay, Binding Assay, Double Staining, Fluorescence

DiI-HDL uptake, DiI-LDL binding, SR-BI immunodetection and Nile Red/filipin double stain in 24, 48 and 96 h cultured 3T3 Swiss mouse fibroblasts. The figure shows DiI-HDL uptake, DiI-LDL binding, SR-BI immunocytofluorescence and merged color images obtained by Nile Red/filipin double staining, that show polar lipids and cytoplasmic membranes (NR polar lipids) in red, LDs (NR neutral lipids) in green/yellow and plasma membrane cholesterol (filipin) in blue. All detections were tested on fibroblasts cultured for 24, 48 and 96 h after contact loss in standard medium. Each experiment was carried out at least twice and the relative graphs and histograms show the mean values ± SE for each single fluorescence emission signal evaluated in at least 200 cells for each group. Bar is 30 μm. *p
Figure Legend Snippet: DiI-HDL uptake, DiI-LDL binding, SR-BI immunodetection and Nile Red/filipin double stain in 24, 48 and 96 h cultured 3T3 Swiss mouse fibroblasts. The figure shows DiI-HDL uptake, DiI-LDL binding, SR-BI immunocytofluorescence and merged color images obtained by Nile Red/filipin double staining, that show polar lipids and cytoplasmic membranes (NR polar lipids) in red, LDs (NR neutral lipids) in green/yellow and plasma membrane cholesterol (filipin) in blue. All detections were tested on fibroblasts cultured for 24, 48 and 96 h after contact loss in standard medium. Each experiment was carried out at least twice and the relative graphs and histograms show the mean values ± SE for each single fluorescence emission signal evaluated in at least 200 cells for each group. Bar is 30 μm. *p

Techniques Used: Binding Assay, Immunodetection, Staining, Cell Culture, Double Staining, Fluorescence

21) Product Images from "Emetine inhibits Zika and Ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry"

Article Title: Emetine inhibits Zika and Ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry

Journal: Cell Discovery

doi: 10.1038/s41421-018-0034-1

Emetine inhibits EBOV infection in vitro and in vivo. a Dose–response curve showing the inhibition effect of emetine treatment on Ebola VLP entry in HeLa cells. b Dose–response curve showing the inhibition effect of emetine treatment on infection of Ebola live virus in Vero E6 cells. c The survival curve of MA-EBOV infected mouse treated with 1 mg/kg emetine every day. Six to eight week-old female BALB/c mice were randomly assigned into groups ( N = 6 animals). All the mice were challenged with a lethal dose of 1000 times the LD 50 mouse adapted EBOV via IP treatments with either emetine (1 mg/kg/day) or PBS (same volume for the control group) were initiated at 3 h before the challenge and continued for up to 6 days post infection. Survival was monitored for 28 days post infection. d Top, dose–response curve showing the effect of emetine on cholesterol accumulation measured by filipin dye fluorescence intensity (IC 50 = 9.03 µM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with filipin (unesterified free cholesterol, green) and nuclear green (nuclei, blue). e Top, dose–response curve showing the effect of emetine on lipid accumulation measured by Nile red fluorescence intensity (IC 50 = 551 nM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with Nile red (lipids, yellow) and Hoechst 33342 (nuclei, blue). f Top, dose–response curve showing the effect of emetine on acidic organelle accumulation measured by LysoTracker dye fluorescence intensity (IC 50 = 26.2 nM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with LysoTracker dye (acidic organelles, red) and Hoechst 33342 (nuclei, blue). All curves represent best fits for calculating the IC 50 values (graph inset). All values represent mean ± SD ( N = 3 replicates). Scale bar=50 µm
Figure Legend Snippet: Emetine inhibits EBOV infection in vitro and in vivo. a Dose–response curve showing the inhibition effect of emetine treatment on Ebola VLP entry in HeLa cells. b Dose–response curve showing the inhibition effect of emetine treatment on infection of Ebola live virus in Vero E6 cells. c The survival curve of MA-EBOV infected mouse treated with 1 mg/kg emetine every day. Six to eight week-old female BALB/c mice were randomly assigned into groups ( N = 6 animals). All the mice were challenged with a lethal dose of 1000 times the LD 50 mouse adapted EBOV via IP treatments with either emetine (1 mg/kg/day) or PBS (same volume for the control group) were initiated at 3 h before the challenge and continued for up to 6 days post infection. Survival was monitored for 28 days post infection. d Top, dose–response curve showing the effect of emetine on cholesterol accumulation measured by filipin dye fluorescence intensity (IC 50 = 9.03 µM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with filipin (unesterified free cholesterol, green) and nuclear green (nuclei, blue). e Top, dose–response curve showing the effect of emetine on lipid accumulation measured by Nile red fluorescence intensity (IC 50 = 551 nM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with Nile red (lipids, yellow) and Hoechst 33342 (nuclei, blue). f Top, dose–response curve showing the effect of emetine on acidic organelle accumulation measured by LysoTracker dye fluorescence intensity (IC 50 = 26.2 nM). Bottom, fluorescence images of fibroblast cells treated with DMSO or emetine and stained with LysoTracker dye (acidic organelles, red) and Hoechst 33342 (nuclei, blue). All curves represent best fits for calculating the IC 50 values (graph inset). All values represent mean ± SD ( N = 3 replicates). Scale bar=50 µm

Techniques Used: Infection, In Vitro, In Vivo, Inhibition, Mouse Assay, Fluorescence, Staining

22) Product Images from "Uptake of Helicobacter pylori Vesicles Is Facilitated by Clathrin-Dependent and Clathrin-Independent Endocytic Pathways"

Article Title: Uptake of Helicobacter pylori Vesicles Is Facilitated by Clathrin-Dependent and Clathrin-Independent Endocytic Pathways

Journal: mBio

doi: 10.1128/mBio.00979-14

Cell membrane cholesterol levels are important for H. pylori vesicle uptake. (A) H. pylori vesicles were incubated with MβCD-treated AGS cells for 20 min. Vesicle internalization decreased to 62% after pretreatment and to 41% when 10 mM MβCD was present during uptake compared to untreated cells (100%). Treatment of AGS cells with 4 mM MβCD did not affect vesicle internalization. Internalization of Tfn was not affected by MβCD. All experiments were performed in triplicate ( n = 2). Statistical significance was as follows: with 10 mM MβCD, P for Ves was 0.0313 and Tfn values were NS; with 10 mM MβCD pretreatment, P for Ves was 0.0313 and Tfn values were NS; with 4 mM MβCD, Ves values and Tfn values were NS. (B) H. pylori vesicles were incubated with filipin-treated AGS cells for 20 min. Vesicle internalization was not affected (105%) relative to untreated cells (100%). Internalization of Tfn was not affected. For Ves, n = 3 (experiments were performed in triplicate); values were NS; for Tfn, n = 2 (experiments were performed in triplicate); values were NS. (C) Electron micrographs of untreated and MβCD-treated H. pylori vesicles show that treated vesicles are intact and have the same morphology as untreated vesicles. Scale bar = 0.5 µm. (D) Biotin linker-labeled vesicles were treated with MβCD or PBS and then incubated with AGS cells for 20 min at 37°C. Reducing conditions allowed detection of intracellular vesicles after staining with Strp-FITC and microscopy analysis of 100 AGS cells. Counting the number of intracellular vesicles revealed no difference in cholesterol-dependent vesicle uptake.
Figure Legend Snippet: Cell membrane cholesterol levels are important for H. pylori vesicle uptake. (A) H. pylori vesicles were incubated with MβCD-treated AGS cells for 20 min. Vesicle internalization decreased to 62% after pretreatment and to 41% when 10 mM MβCD was present during uptake compared to untreated cells (100%). Treatment of AGS cells with 4 mM MβCD did not affect vesicle internalization. Internalization of Tfn was not affected by MβCD. All experiments were performed in triplicate ( n = 2). Statistical significance was as follows: with 10 mM MβCD, P for Ves was 0.0313 and Tfn values were NS; with 10 mM MβCD pretreatment, P for Ves was 0.0313 and Tfn values were NS; with 4 mM MβCD, Ves values and Tfn values were NS. (B) H. pylori vesicles were incubated with filipin-treated AGS cells for 20 min. Vesicle internalization was not affected (105%) relative to untreated cells (100%). Internalization of Tfn was not affected. For Ves, n = 3 (experiments were performed in triplicate); values were NS; for Tfn, n = 2 (experiments were performed in triplicate); values were NS. (C) Electron micrographs of untreated and MβCD-treated H. pylori vesicles show that treated vesicles are intact and have the same morphology as untreated vesicles. Scale bar = 0.5 µm. (D) Biotin linker-labeled vesicles were treated with MβCD or PBS and then incubated with AGS cells for 20 min at 37°C. Reducing conditions allowed detection of intracellular vesicles after staining with Strp-FITC and microscopy analysis of 100 AGS cells. Counting the number of intracellular vesicles revealed no difference in cholesterol-dependent vesicle uptake.

Techniques Used: Incubation, Labeling, Staining, Microscopy

23) Product Images from "Small GTPase Rab14 down-regulates UT-A1 urea transport activity through enhanced clathrin-dependent endocytosis"

Article Title: Small GTPase Rab14 down-regulates UT-A1 urea transport activity through enhanced clathrin-dependent endocytosis

Journal: The FASEB Journal

doi: 10.1096/fj.13-229294

Inhibition of clathrin endocytic pathway blocks Rab14-induced UT-A1 membrane reduction. A ) Cell surface biotinylation assay. UT-A1-HEK 293 cells were transfected with or without Rab14. After 48 h, cells were treated with 10 μg/ml chlorpromazine or 1 μM filipin for 1 h, and then processed for biotinylation. The cells were lysed, and the biotinylated samples and total proteins were analyzed by Western blot with antibodies to UT-A1, Rab14 (HA) or actin. B ) Chlorpromazine treatment. External FLAG-tagged UT-A1 HEK 293 cells were transfected with pcDNA3-HARab14 for 48 h. Cells were treated with or without 10 μg/ml chlorpromazine for 1 h. Cell surface UT-A1 was labeled with anti-FLAG antibody on ice for 1 h. After washing, the cells were rewarmed at 37°C for 30 min. Cells were then fixed, and the UT-A1 remaining on the cell surface was detected by Texas Red-conjugated anti-mouse antibody without membrane permeabilization. C ) Knocking down μ2 by siRNA. External FLAG-tagged UT-A1 HEK 293 cells were cotransfected with Rab14 together with pSuppressor-scramble or pSuppressor-μ2 for 48 h. UT-A1 internalization was induced, and cell surface UT-A1 was detected as described above.
Figure Legend Snippet: Inhibition of clathrin endocytic pathway blocks Rab14-induced UT-A1 membrane reduction. A ) Cell surface biotinylation assay. UT-A1-HEK 293 cells were transfected with or without Rab14. After 48 h, cells were treated with 10 μg/ml chlorpromazine or 1 μM filipin for 1 h, and then processed for biotinylation. The cells were lysed, and the biotinylated samples and total proteins were analyzed by Western blot with antibodies to UT-A1, Rab14 (HA) or actin. B ) Chlorpromazine treatment. External FLAG-tagged UT-A1 HEK 293 cells were transfected with pcDNA3-HARab14 for 48 h. Cells were treated with or without 10 μg/ml chlorpromazine for 1 h. Cell surface UT-A1 was labeled with anti-FLAG antibody on ice for 1 h. After washing, the cells were rewarmed at 37°C for 30 min. Cells were then fixed, and the UT-A1 remaining on the cell surface was detected by Texas Red-conjugated anti-mouse antibody without membrane permeabilization. C ) Knocking down μ2 by siRNA. External FLAG-tagged UT-A1 HEK 293 cells were cotransfected with Rab14 together with pSuppressor-scramble or pSuppressor-μ2 for 48 h. UT-A1 internalization was induced, and cell surface UT-A1 was detected as described above.

Techniques Used: Inhibition, Cell Surface Biotinylation Assay, Transfection, Western Blot, Labeling

24) Product Images from "Nanomaterials can Dynamically Steer Cell Responses to Biological Ligands"

Article Title: Nanomaterials can Dynamically Steer Cell Responses to Biological Ligands

Journal: Small (Weinheim an Der Bergstrasse, Germany)

doi: 10.1002/smll.201001518

(A) Single keratinocyte cell migration was quantified on substrates adsorbed with ligand alone, nanocarriers alone, and ligand-conjugated nanocarriers over a range of varying ANC sizes and ligand concentrations. Data represents the average of all data from 3 experiments performed in duplicate. For each experiment, n=60 tracks. Error bars represent standard error around the mean. At comparable net levels of ligands presented, the smaller sized nanocarriers (30nm) elicited higher levels of cell motility than the larger sized nanocarriers (100nm). (B) We examined whether the effects of ANC carriers on cell migration were due to the differential exposure and bioactivity of ligands from nanocarriers of different sizes. ELISA techniques were used to quantify the exposure of cell binding domains of the nanocarrier-functionalized type III-9–10 fibronectin fragment. While increased ligand bioactivity (cell binding domain exposure) generally correlated with increased cell migration speed, it did not uniquely govern cell speeds; additionally, geometric (size) aspects of ANCs additionally modulated cell migration. (C) The role of ligand-nanocarrier endocytosis on cell migration was examined by comparing cell migration on physisorbed ligand-ANC (“passive” condition) versus ligand-ANCs controls chemically immobilized via oxygen plasma treatment of well plates. Cells were pretreated with either filipin (caveolin inhibitor) or chlorpromazine (clathrin inhibitor) before deposition on passively adsorbed substrates. Images were acquired and analyzed as described above. Cell migration on physisorbed ligand-ANCs was significantly enhanced in comparison to that on immobilized substrates. Inhibition of clathrin but not caveolin reduced levels of cell migration indicating a key role for clathrin mediated endocytosis.
Figure Legend Snippet: (A) Single keratinocyte cell migration was quantified on substrates adsorbed with ligand alone, nanocarriers alone, and ligand-conjugated nanocarriers over a range of varying ANC sizes and ligand concentrations. Data represents the average of all data from 3 experiments performed in duplicate. For each experiment, n=60 tracks. Error bars represent standard error around the mean. At comparable net levels of ligands presented, the smaller sized nanocarriers (30nm) elicited higher levels of cell motility than the larger sized nanocarriers (100nm). (B) We examined whether the effects of ANC carriers on cell migration were due to the differential exposure and bioactivity of ligands from nanocarriers of different sizes. ELISA techniques were used to quantify the exposure of cell binding domains of the nanocarrier-functionalized type III-9–10 fibronectin fragment. While increased ligand bioactivity (cell binding domain exposure) generally correlated with increased cell migration speed, it did not uniquely govern cell speeds; additionally, geometric (size) aspects of ANCs additionally modulated cell migration. (C) The role of ligand-nanocarrier endocytosis on cell migration was examined by comparing cell migration on physisorbed ligand-ANC (“passive” condition) versus ligand-ANCs controls chemically immobilized via oxygen plasma treatment of well plates. Cells were pretreated with either filipin (caveolin inhibitor) or chlorpromazine (clathrin inhibitor) before deposition on passively adsorbed substrates. Images were acquired and analyzed as described above. Cell migration on physisorbed ligand-ANCs was significantly enhanced in comparison to that on immobilized substrates. Inhibition of clathrin but not caveolin reduced levels of cell migration indicating a key role for clathrin mediated endocytosis.

Techniques Used: Migration, Enzyme-linked Immunosorbent Assay, Binding Assay, Inhibition

25) Product Images from "Neuronal and epithelial cell rescue resolves chronic systemic inflammation in the lipid storage disorder Niemann-Pick C"

Article Title: Neuronal and epithelial cell rescue resolves chronic systemic inflammation in the lipid storage disorder Niemann-Pick C

Journal: Human Molecular Genetics

doi: 10.1093/hmg/dds126

Macrophages respond locally to epithelial cell rescue. ( A ) Dox administered to P65 or older R; N; Npc1 −/− mice produced a mosaic rescue effect in liver tissue. Regions (inside semi-circle) that did not produce NPC1-YFP immunofluorescence (GFP, green) remained cholesterol positive (Filipin, blue) and had focal accumulation (arrow) of clustered macrophages (CD68, red). ( B ) In lung alveoli, foam cell CD68-positive macrophages (red) were seen proximal to simple squamous epithelial cells (top panel). Macrophages near simple squamous epithelial cells producing NPC1-YFP (bottom panel, yellow arrow) were reduced in size in comparison to the large foam macrophages surrounding non-NPC1-YFP-positive epithelial regions. ( C ) Apart from a few sparse squamous cells, NPC1-YFP immunofluorescence predominated in the epithelial linings (arrow) of bronchioles in R; N mice given Dox. ( D ) Quantification of the percent area that was CD68 positive in alveoli shows a significant ( P = 0.0061, n = 3 mice) decrease in CD68 in alveoli regions most proximal to bronchioles. Scale bars are 50 µm.
Figure Legend Snippet: Macrophages respond locally to epithelial cell rescue. ( A ) Dox administered to P65 or older R; N; Npc1 −/− mice produced a mosaic rescue effect in liver tissue. Regions (inside semi-circle) that did not produce NPC1-YFP immunofluorescence (GFP, green) remained cholesterol positive (Filipin, blue) and had focal accumulation (arrow) of clustered macrophages (CD68, red). ( B ) In lung alveoli, foam cell CD68-positive macrophages (red) were seen proximal to simple squamous epithelial cells (top panel). Macrophages near simple squamous epithelial cells producing NPC1-YFP (bottom panel, yellow arrow) were reduced in size in comparison to the large foam macrophages surrounding non-NPC1-YFP-positive epithelial regions. ( C ) Apart from a few sparse squamous cells, NPC1-YFP immunofluorescence predominated in the epithelial linings (arrow) of bronchioles in R; N mice given Dox. ( D ) Quantification of the percent area that was CD68 positive in alveoli shows a significant ( P = 0.0061, n = 3 mice) decrease in CD68 in alveoli regions most proximal to bronchioles. Scale bars are 50 µm.

Techniques Used: Mouse Assay, Produced, Immunofluorescence

26) Product Images from "Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway"

Article Title: Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway

Journal: Wellcome open research

doi: 10.12688/wellcomeopenres.10036.1

Certain NPC therapeutics promote clearance of intracellular mycobacteria. (A) Cholesterol distribution in wild-type RAW 264.7 macrophages treated with U18666A (2μg/ml) for 48h and subsequently treated with either vehicle (DMSO), curcumin (30μM/24h), miglustat (50μM/72h) or hydroxypropryl-β-cyclodextrin (250μM/24h). Blue, filipin (cholesterol). Scale bar, 5μm. (B) Correlation between extent of infection with mCherry-expressing BCG and levels of RAW 264.7 fluorescence as quantified using flow cytometry (i) Representative histograms showing fluorescence of RAW 264.7 cell cultures infected with BCG at low or high MOI (10 or 100 respectively). (ii) Fluorescence of RAW 264.7 cell cultures infected with BCG at low or high MOI (10,000 cells counted). Mean ± SEM, N=4. (C) Effect of curcumin on intracellular BCG levels in RAW 264.7 macrophages. Fold change in mean fluorescence of RAW 264.7 macrophages after 48h infection with mCherry-BCG (MOI, 12.5) and subsequent treatment with curcumin (30μM; 24h), miglustat (50μM; 72h), combined curcumin (30μM; 24h) and miglustat (50μM; 72h) or hydroxypropyl-β-cyclodextrin (250μM; 24h). Fold change in fluorescence given relative to untreated, infected control. Mean ± SEM. N=4. * p
Figure Legend Snippet: Certain NPC therapeutics promote clearance of intracellular mycobacteria. (A) Cholesterol distribution in wild-type RAW 264.7 macrophages treated with U18666A (2μg/ml) for 48h and subsequently treated with either vehicle (DMSO), curcumin (30μM/24h), miglustat (50μM/72h) or hydroxypropryl-β-cyclodextrin (250μM/24h). Blue, filipin (cholesterol). Scale bar, 5μm. (B) Correlation between extent of infection with mCherry-expressing BCG and levels of RAW 264.7 fluorescence as quantified using flow cytometry (i) Representative histograms showing fluorescence of RAW 264.7 cell cultures infected with BCG at low or high MOI (10 or 100 respectively). (ii) Fluorescence of RAW 264.7 cell cultures infected with BCG at low or high MOI (10,000 cells counted). Mean ± SEM, N=4. (C) Effect of curcumin on intracellular BCG levels in RAW 264.7 macrophages. Fold change in mean fluorescence of RAW 264.7 macrophages after 48h infection with mCherry-BCG (MOI, 12.5) and subsequent treatment with curcumin (30μM; 24h), miglustat (50μM; 72h), combined curcumin (30μM; 24h) and miglustat (50μM; 72h) or hydroxypropyl-β-cyclodextrin (250μM; 24h). Fold change in fluorescence given relative to untreated, infected control. Mean ± SEM. N=4. * p

Techniques Used: Infection, Expressing, Fluorescence, Flow Cytometry, Cytometry

Mycobacterial cell wall lipids induce NPC phenotypes in the absence of live mycobacteria. (A) General structure of mycolic acids. The lipid consists of a mycolic motif (with alkyl chain of variable length) and meromycolate chain with a distal and proximal functional group (X/Y) (adapted from 74 ). (B) Cholesterol storage in RAW 264.7 macrophages treated with BCG MAMES/FAMES (18h treatment). Blue, filipin (cholesterol). Scale bar, 5μm. (C) (i) Trafficking of GM1 ganglioside in RAW 264.7 macrophages treated with MAMES/FAMES. Red, cholera toxin subunit B (GM1 ganglioside); blue, Hoescht 33258 (nucleus). (ii) Sphingomyelin distribution in RAW 264.7 macrophages treated with MAMES/FAMES. Green, lysenin (sphingomyelin); blue, Hoescht 33258 (nucleus). (MOI 12.5; 5μg/ml). Scale bar, 5μm. (D) Trafficking of GM1 ganglioside in (i) untreated RAW 264.7 macrophages, post 18h incubation with secreted BCG lipids (ii) or heat-treated lipids (iii). Red, cholera toxin subunit B (GM1 gangliosides); blue, Hoescht 33258 (nucleus). (E) (i) Cholesterol storage in RAW 264.7 macrophages treated with BCG mycolic acids. Blue, filipin (cholesterol); red, propidium iodide (nucleus). (ii) Sphingomyelin distribution in RAW 264.7 macrophages treated with BCG mycolic acids. Red, lysenin (sphingomyelin); blue Hoescht 33258 (nucleus) (24h treatment; 5μg/ml). Scale bar, 5μm. (F) (i) Cholesterol storage in primary human macrophages treated with BCG mycolic acids. Blue, filipin (cholesterol) (ii) Trafficking of GM1 ganglioside in primary human macrophages treated with BCG mycolic acids. Green, cholera toxin subunit B (GM1 ganglioside) (24h treatment; 5μg/ml). Scale bar, 5μm. (G) LysoTracker staining of RAW 264.7 macrophages 24h post incubation with purified mycolic acid esters (glycomycolates; 1ng/ml). Mean ± SEM. N=4. * p
Figure Legend Snippet: Mycobacterial cell wall lipids induce NPC phenotypes in the absence of live mycobacteria. (A) General structure of mycolic acids. The lipid consists of a mycolic motif (with alkyl chain of variable length) and meromycolate chain with a distal and proximal functional group (X/Y) (adapted from 74 ). (B) Cholesterol storage in RAW 264.7 macrophages treated with BCG MAMES/FAMES (18h treatment). Blue, filipin (cholesterol). Scale bar, 5μm. (C) (i) Trafficking of GM1 ganglioside in RAW 264.7 macrophages treated with MAMES/FAMES. Red, cholera toxin subunit B (GM1 ganglioside); blue, Hoescht 33258 (nucleus). (ii) Sphingomyelin distribution in RAW 264.7 macrophages treated with MAMES/FAMES. Green, lysenin (sphingomyelin); blue, Hoescht 33258 (nucleus). (MOI 12.5; 5μg/ml). Scale bar, 5μm. (D) Trafficking of GM1 ganglioside in (i) untreated RAW 264.7 macrophages, post 18h incubation with secreted BCG lipids (ii) or heat-treated lipids (iii). Red, cholera toxin subunit B (GM1 gangliosides); blue, Hoescht 33258 (nucleus). (E) (i) Cholesterol storage in RAW 264.7 macrophages treated with BCG mycolic acids. Blue, filipin (cholesterol); red, propidium iodide (nucleus). (ii) Sphingomyelin distribution in RAW 264.7 macrophages treated with BCG mycolic acids. Red, lysenin (sphingomyelin); blue Hoescht 33258 (nucleus) (24h treatment; 5μg/ml). Scale bar, 5μm. (F) (i) Cholesterol storage in primary human macrophages treated with BCG mycolic acids. Blue, filipin (cholesterol) (ii) Trafficking of GM1 ganglioside in primary human macrophages treated with BCG mycolic acids. Green, cholera toxin subunit B (GM1 ganglioside) (24h treatment; 5μg/ml). Scale bar, 5μm. (G) LysoTracker staining of RAW 264.7 macrophages 24h post incubation with purified mycolic acid esters (glycomycolates; 1ng/ml). Mean ± SEM. N=4. * p

Techniques Used: Functional Assay, Incubation, Staining, Purification

27) Product Images from "Characterization of Gaucher disease bone marrow mesenchymal stromal cells reveals an altered inflammatory secretome"

Article Title: Characterization of Gaucher disease bone marrow mesenchymal stromal cells reveals an altered inflammatory secretome

Journal: Blood

doi: 10.1182/blood-2009-02-205708

Cholesterol content and lactosylceramide trafficking . (A) MSCs stained with filipin and observed under a fluorescence microscope; scale bar = 20 μm. (B) Total cholesterol and cholesteryl ester content expressed as nanograms per milligrams of total
Figure Legend Snippet: Cholesterol content and lactosylceramide trafficking . (A) MSCs stained with filipin and observed under a fluorescence microscope; scale bar = 20 μm. (B) Total cholesterol and cholesteryl ester content expressed as nanograms per milligrams of total

Techniques Used: Staining, Fluorescence, Microscopy

28) Product Images from "Therapeutics to Promote CNS Repair: A Natural Human Neuron-Binding IgM Regulates Membrane-Raft Dynamics and Improves Motility in a Mouse Model of Multiple Sclerosis"

Article Title: Therapeutics to Promote CNS Repair: A Natural Human Neuron-Binding IgM Regulates Membrane-Raft Dynamics and Improves Motility in a Mouse Model of Multiple Sclerosis

Journal: Journal of clinical immunology

doi: 10.1007/s10875-012-9795-8

rHIgM12 clusters membrane rafts on neuronal membranes A. DIV1 hippocampal neurons were fixed and stained with rHIgM12. B – C . Live DIV3 hippocampal neurons were treated with rHIgM12 at 37°C for 30 min, then fixed without permeabilization and stained with anti-human IgM secondary antibody, filipin ( B , Cholesterol), or cholera toxin B ( C , GM1) that labeled cholesterol or ganglioside GM1, respectively. rHIgM12 aggregated on neuronal surfaces ( B1–C1 ) and induced the clustering of cholesterol ( B2 ) and GM1 ( C2 ). b1–b2 and c1–c2 show a higher magnification of boxed regions in B and C . Arrow heads indicate the localization of both aggregated rHIgM12 and cholesterol or GM1. Scale bar, 10 μm.
Figure Legend Snippet: rHIgM12 clusters membrane rafts on neuronal membranes A. DIV1 hippocampal neurons were fixed and stained with rHIgM12. B – C . Live DIV3 hippocampal neurons were treated with rHIgM12 at 37°C for 30 min, then fixed without permeabilization and stained with anti-human IgM secondary antibody, filipin ( B , Cholesterol), or cholera toxin B ( C , GM1) that labeled cholesterol or ganglioside GM1, respectively. rHIgM12 aggregated on neuronal surfaces ( B1–C1 ) and induced the clustering of cholesterol ( B2 ) and GM1 ( C2 ). b1–b2 and c1–c2 show a higher magnification of boxed regions in B and C . Arrow heads indicate the localization of both aggregated rHIgM12 and cholesterol or GM1. Scale bar, 10 μm.

Techniques Used: Staining, Labeling

29) Product Images from "Role of Exchange Protein Activated by cAMP 1 in Regulating Rates of Microtubule Formation in Cystic Fibrosis Epithelial Cells"

Article Title: Role of Exchange Protein Activated by cAMP 1 in Regulating Rates of Microtubule Formation in Cystic Fibrosis Epithelial Cells

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2014-0462OC

Effect of EPAC1-selective agonist, 8-cpt-cA, on cholesterol distribution in IB3 cells. ( A ) IB3 cells were either treated with vehicle or with 25 μM 8-cpt-cA for 24 hours and assessed for cholesterol by filipin staining. To ensure efficacy of 8-cpt-cA, cells were immunostained with phospho-AKT (pAKT) antibodies and 4′,6-diamidino-2-phenylindole. Representative images are shown from four separate experiments. ( B ) Filipin imaging was quantified by assessing cells for perinuclear cholesterol accumulation by a grader blinded to experimental group. Data are presented as percentage of cells with perinuclear accumulation. IB3 cells exhibited 82.3 (±3.2)% of cells with accumulation compared with 23.4 (±6.1)% of IB3 cells treated with 8-cpt-cA ( n = 20 separate images for each condition; * P
Figure Legend Snippet: Effect of EPAC1-selective agonist, 8-cpt-cA, on cholesterol distribution in IB3 cells. ( A ) IB3 cells were either treated with vehicle or with 25 μM 8-cpt-cA for 24 hours and assessed for cholesterol by filipin staining. To ensure efficacy of 8-cpt-cA, cells were immunostained with phospho-AKT (pAKT) antibodies and 4′,6-diamidino-2-phenylindole. Representative images are shown from four separate experiments. ( B ) Filipin imaging was quantified by assessing cells for perinuclear cholesterol accumulation by a grader blinded to experimental group. Data are presented as percentage of cells with perinuclear accumulation. IB3 cells exhibited 82.3 (±3.2)% of cells with accumulation compared with 23.4 (±6.1)% of IB3 cells treated with 8-cpt-cA ( n = 20 separate images for each condition; * P

Techniques Used: Cycling Probe Technology, Staining, Imaging

30) Product Images from "Lipid Raft-Based Membrane Compartmentation of a Plant Transport Protein Expressed in Saccharomyces cerevisiae"

Article Title: Lipid Raft-Based Membrane Compartmentation of a Plant Transport Protein Expressed in Saccharomyces cerevisiae

Journal: Eukaryotic Cell

doi: 10.1128/EC.00206-05

HUP1-GFP expressed in Schizosaccharomyces pombe (FY254/pHUP1-GFP). Sterol-rich membrane domains were visualized with filipin (A; fluorescence microscope, UV light) and HUP1-GFP localization (B; confocal microscope). Size bars, 10 μm.
Figure Legend Snippet: HUP1-GFP expressed in Schizosaccharomyces pombe (FY254/pHUP1-GFP). Sterol-rich membrane domains were visualized with filipin (A; fluorescence microscope, UV light) and HUP1-GFP localization (B; confocal microscope). Size bars, 10 μm.

Techniques Used: Fluorescence, Microscopy

31) Product Images from "The Sur7 Protein Regulates Plasma Membrane Organization and Prevents Intracellular Cell Wall Growth in Candida albicans"

Article Title: The Sur7 Protein Regulates Plasma Membrane Organization and Prevents Intracellular Cell Wall Growth in Candida albicans

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E08-05-0479

Hyphal morphogenesis defects of sur7 Δ mutant. (A) Calcofluor staining of hyphal cells induced with serum for 75 min. (B) Filipin staining of cells induced with 100 mM GlcNAc for 70 min to form hyphae. (C) Cells were spotted on solid medium agar
Figure Legend Snippet: Hyphal morphogenesis defects of sur7 Δ mutant. (A) Calcofluor staining of hyphal cells induced with serum for 75 min. (B) Filipin staining of cells induced with 100 mM GlcNAc for 70 min to form hyphae. (C) Cells were spotted on solid medium agar

Techniques Used: Mutagenesis, Staining

32) Product Images from "Oxidized Low-Density Lipoprotein Is Present in Astrocytes Surrounding Cerebral Infarcts and Stimulates Astrocyte Interleukin-6 Secretion"

Article Title: Oxidized Low-Density Lipoprotein Is Present in Astrocytes Surrounding Cerebral Infarcts and Stimulates Astrocyte Interleukin-6 Secretion

Journal: The American Journal of Pathology

doi:

Co-localization of oxLDL epitope, DiI label, and cholesterol in astrocytes exposed to DiI-oxLDL-L ( A ) or DiI-LDL ( B ) for 24 hours. Antibody AB3232 (blue) and filipin (green) were used to localize oxLDL epitope and cholesterol, respectively. DiI in these images is red. Purple/pink depicts co-localization of DiI and oxLDL. White depicts co-localization of all three molecules. A: OxLDL immunoreactivity co-localized with DiI and the majority of filipin staining in astrocytes exposed to DiI-oxLDL-L. B: DiI predominantly co-localized with filipin in cells exposed to DiI-LDL. There was also a small amount of immunoreactivity for oxLDL; however, this was not greater than background levels.
Figure Legend Snippet: Co-localization of oxLDL epitope, DiI label, and cholesterol in astrocytes exposed to DiI-oxLDL-L ( A ) or DiI-LDL ( B ) for 24 hours. Antibody AB3232 (blue) and filipin (green) were used to localize oxLDL epitope and cholesterol, respectively. DiI in these images is red. Purple/pink depicts co-localization of DiI and oxLDL. White depicts co-localization of all three molecules. A: OxLDL immunoreactivity co-localized with DiI and the majority of filipin staining in astrocytes exposed to DiI-oxLDL-L. B: DiI predominantly co-localized with filipin in cells exposed to DiI-LDL. There was also a small amount of immunoreactivity for oxLDL; however, this was not greater than background levels.

Techniques Used: Staining

33) Product Images from "TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells"

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200420773

Lipid rafts are involved in clustering of receptors and signaling. ( A ) Confocal z-section images demonstrate caveolin-1 labeled with Alexa Fluor 594 (red) and GM1, identified with cholera toxin β-subunit (CTB) conjugated to Alexa Fluor 488 (green), on the apical surfaces of polarized 16HBE cells permeabilized after stimulation with Pam 3 Cys-Ser-Lys 4 . ( B ) CF nasal polyp cells were infected with P. aeruginosa PAO1 (PA) and grown on semipermeable supports, and transmission electron micrograph were obtained after 3 hours of bacterial exposure. Arrow (Cav) indicates the flask-shaped electron-dense structures typical of caveolae (magnification, ∞30,000). ( C ) Flow cytometry was used to detect superficial caveolin-1 on polarized 16HBE cells after exposure to S. aureus . Unstim, unstimulated. ( D ) Aliquots of Triton-insoluble lysates of 16HBE cells obtained before (–) and after (+) stimulation with P. aeruginosa PAO1 were fractionated on discontinuous sucrose gradients (4–40%) and were immunoblotted with anti-flotillin, anti-caveolin, anti-TLR-2, anti-IRAK-1, or anti-asialoGM1. Downward arrow indicates raft fraction containing all the components after stimulation. ( E ) Aliquots of sucrose gradient fractions from cells treated with filipin prior to stimulation with P. aeruginosa PAO1 were immunoblotted with anti-flotillin, anti-TLR2, and anti-asialoGM1.
Figure Legend Snippet: Lipid rafts are involved in clustering of receptors and signaling. ( A ) Confocal z-section images demonstrate caveolin-1 labeled with Alexa Fluor 594 (red) and GM1, identified with cholera toxin β-subunit (CTB) conjugated to Alexa Fluor 488 (green), on the apical surfaces of polarized 16HBE cells permeabilized after stimulation with Pam 3 Cys-Ser-Lys 4 . ( B ) CF nasal polyp cells were infected with P. aeruginosa PAO1 (PA) and grown on semipermeable supports, and transmission electron micrograph were obtained after 3 hours of bacterial exposure. Arrow (Cav) indicates the flask-shaped electron-dense structures typical of caveolae (magnification, ∞30,000). ( C ) Flow cytometry was used to detect superficial caveolin-1 on polarized 16HBE cells after exposure to S. aureus . Unstim, unstimulated. ( D ) Aliquots of Triton-insoluble lysates of 16HBE cells obtained before (–) and after (+) stimulation with P. aeruginosa PAO1 were fractionated on discontinuous sucrose gradients (4–40%) and were immunoblotted with anti-flotillin, anti-caveolin, anti-TLR-2, anti-IRAK-1, or anti-asialoGM1. Downward arrow indicates raft fraction containing all the components after stimulation. ( E ) Aliquots of sucrose gradient fractions from cells treated with filipin prior to stimulation with P. aeruginosa PAO1 were immunoblotted with anti-flotillin, anti-TLR2, and anti-asialoGM1.

Techniques Used: Labeling, CtB Assay, Infection, Transmission Assay, Flow Cytometry, Cytometry

34) Product Images from "The multivesicular body is the major internal site of prion conversion"

Article Title: The multivesicular body is the major internal site of prion conversion

Journal: Journal of Cell Science

doi: 10.1242/jcs.165472

Knocking down Hrs or Tsg101 alters PrPsc localization and decreases the level of PrPsc. (A) Hrs or Tsg101 was knocked down (KD) for the indicated times before immunostaining. The merge image shows PrPsc (red), LAMP1 (green) and EEA1 (blue). (B) Western blot of PrPsc and PrPc in lysates from control and in cells knocked down for the following proteins: Rab7a, Hrs, Tsg101. (C) Western blot of PrPsc in lysates from SMB cells depleted of Alix. (D) Western blot of PrPsc in lysates prepared from SMB cells stably expressing the indicated Rab constructs. Immunoblots were probed with antibodies against PrP and actin. (E) Filipin staining of SMB cells grown under different conditions. Control cells, cells incubated in U18666A (50 µM final concentration) for 2 days, Rab7a-knockdown cells, and Tsg101-knockdown cells were stained with filipin and anti-LAMP1 antibody. Scale bar, 10 µm. (F) Relative cholesterol levels under different conditions. Data (mean±s.d., n = 10) were normalized by setting the intensity of the background and U18666A-treated cells to 0% and 100%, respectively.
Figure Legend Snippet: Knocking down Hrs or Tsg101 alters PrPsc localization and decreases the level of PrPsc. (A) Hrs or Tsg101 was knocked down (KD) for the indicated times before immunostaining. The merge image shows PrPsc (red), LAMP1 (green) and EEA1 (blue). (B) Western blot of PrPsc and PrPc in lysates from control and in cells knocked down for the following proteins: Rab7a, Hrs, Tsg101. (C) Western blot of PrPsc in lysates from SMB cells depleted of Alix. (D) Western blot of PrPsc in lysates prepared from SMB cells stably expressing the indicated Rab constructs. Immunoblots were probed with antibodies against PrP and actin. (E) Filipin staining of SMB cells grown under different conditions. Control cells, cells incubated in U18666A (50 µM final concentration) for 2 days, Rab7a-knockdown cells, and Tsg101-knockdown cells were stained with filipin and anti-LAMP1 antibody. Scale bar, 10 µm. (F) Relative cholesterol levels under different conditions. Data (mean±s.d., n = 10) were normalized by setting the intensity of the background and U18666A-treated cells to 0% and 100%, respectively.

Techniques Used: Immunostaining, Western Blot, Stable Transfection, Expressing, Construct, Staining, Incubation, Concentration Assay

35) Product Images from "INCREASED MEMBRANE CHOLESTEROL MIGHT RENDER MATURE HIPPOCAMPAL NEURONS MORE SUSCEPTIBLE TO BETA-AMYLOID-INDUCED CALPAIN ACTIVATION AND TAU TOXICITY"

Article Title: INCREASED MEMBRANE CHOLESTEROL MIGHT RENDER MATURE HIPPOCAMPAL NEURONS MORE SUSCEPTIBLE TO BETA-AMYLOID-INDUCED CALPAIN ACTIVATION AND TAU TOXICITY

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.0862-09.2009

Membrane cholesterol levels increased as hippocampal neurons developed in culture (A B) Filipin labeling of 7 (A) and 21 (B) days in culture hippocampal neurons showed intense fluorescence in the soma (s) of neurons in both age groups. The staining extended further into the processes (p) of the older neurons when compared to the younger ones. (C) Quantitative analysis of the relative filipin fluorescence intensity in 7 (white bars) and 21 (black bars) days in culture hippocampal neurons. Note the increased filipin intensity in the soma (s) and processes (p) of 21 days in culture neurons as compared to those of 7 days in culture ones. (D) Cholesterol quantification in cytosol, membrane, and whole cell fractions of neurons cultured for 7 (white bars) and 21 (black bars) days by means of Amplex Red analysis. Values were normalized to total protein in these lysates. Each graph displays the mean values ± S.E.M. and a statistical comparison between 7 and 21 days in culture cells. *Differs from 7 days in culture values, P
Figure Legend Snippet: Membrane cholesterol levels increased as hippocampal neurons developed in culture (A B) Filipin labeling of 7 (A) and 21 (B) days in culture hippocampal neurons showed intense fluorescence in the soma (s) of neurons in both age groups. The staining extended further into the processes (p) of the older neurons when compared to the younger ones. (C) Quantitative analysis of the relative filipin fluorescence intensity in 7 (white bars) and 21 (black bars) days in culture hippocampal neurons. Note the increased filipin intensity in the soma (s) and processes (p) of 21 days in culture neurons as compared to those of 7 days in culture ones. (D) Cholesterol quantification in cytosol, membrane, and whole cell fractions of neurons cultured for 7 (white bars) and 21 (black bars) days by means of Amplex Red analysis. Values were normalized to total protein in these lysates. Each graph displays the mean values ± S.E.M. and a statistical comparison between 7 and 21 days in culture cells. *Differs from 7 days in culture values, P

Techniques Used: Labeling, Fluorescence, Staining, Cell Culture

36) Product Images from "The Importance of the Stem Cell Marker Prominin-1/CD133 in the Uptake of Transferrin and in Iron Metabolism in Human Colon Cancer Caco-2 Cells"

Article Title: The Importance of the Stem Cell Marker Prominin-1/CD133 in the Uptake of Transferrin and in Iron Metabolism in Human Colon Cancer Caco-2 Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025515

Implication of CD133 in endocytosis mainly involved the clathrin pathway. A) Consequences of CD133-specific siRNA knockdown for the effectiveness of chemical modulators of known endocytic pathways in modulating Tf uptake by non-differentiated Caco-2 cells. After treatment with either vehicle alone (control), filipin, chlorpromazine, DMA or MβCD added with lovastatin (MβCD), CD133 high (control siRNA) and CD133 low (CD133 siRNA) non-differentiated Caco-2 cells were exposed to 5 µg/mL Tf-Alexa 488. Cellular internalization of Tf-Alexa 488 was then monitored by flow cytometry. Results are expressed as a % of Tf-Alexa 488 amounts that were internalized in the vehicle treated control. Note the absence of effect of CD133-siRNA knockdown on the major inhibition of Tf-uptake caused by chlorpromazine. Note also the reduced up regulatory effect of cholesterol extraction in the CD133 low situation (MβCD). Data represented mean ± s.e.m. of a triplicate obtained from one representative experiment that was reproduced twice. Comparisons with control: Dunnett's test, *p
Figure Legend Snippet: Implication of CD133 in endocytosis mainly involved the clathrin pathway. A) Consequences of CD133-specific siRNA knockdown for the effectiveness of chemical modulators of known endocytic pathways in modulating Tf uptake by non-differentiated Caco-2 cells. After treatment with either vehicle alone (control), filipin, chlorpromazine, DMA or MβCD added with lovastatin (MβCD), CD133 high (control siRNA) and CD133 low (CD133 siRNA) non-differentiated Caco-2 cells were exposed to 5 µg/mL Tf-Alexa 488. Cellular internalization of Tf-Alexa 488 was then monitored by flow cytometry. Results are expressed as a % of Tf-Alexa 488 amounts that were internalized in the vehicle treated control. Note the absence of effect of CD133-siRNA knockdown on the major inhibition of Tf-uptake caused by chlorpromazine. Note also the reduced up regulatory effect of cholesterol extraction in the CD133 low situation (MβCD). Data represented mean ± s.e.m. of a triplicate obtained from one representative experiment that was reproduced twice. Comparisons with control: Dunnett's test, *p

Techniques Used: Flow Cytometry, Cytometry, Inhibition

37) Product Images from "Selective inhibition of Ebola entry with selective estrogen receptor modulators by disrupting the endolysosomal calcium"

Article Title: Selective inhibition of Ebola entry with selective estrogen receptor modulators by disrupting the endolysosomal calcium

Journal: Scientific Reports

doi: 10.1038/srep41226

Equal dosages of SERMs inhibit Ebola pseudovirion entry and induce cholesterol accumulation. ( a ) Dose-response inhibition of Ebola entry for tamoxifen, clomiphene and U18666a. Data are expressed as the means ± SEM (n = 3). ( b ) Dose-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. ( b ) Time-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. Black arrows indicate examples of cholesterol accumulation. ( d,e ) The quantification of cholesterol accumulation stained with filipin. Data are expressed as the means ± SEM (n ≥ 4). Significant differences versus control group (0 μM or 0 h) are presented by asterisks (*), * P
Figure Legend Snippet: Equal dosages of SERMs inhibit Ebola pseudovirion entry and induce cholesterol accumulation. ( a ) Dose-response inhibition of Ebola entry for tamoxifen, clomiphene and U18666a. Data are expressed as the means ± SEM (n = 3). ( b ) Dose-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. ( b ) Time-response cholesterol accumulation for tamoxifen, clomiphene and U18666a. Black arrows indicate examples of cholesterol accumulation. ( d,e ) The quantification of cholesterol accumulation stained with filipin. Data are expressed as the means ± SEM (n ≥ 4). Significant differences versus control group (0 μM or 0 h) are presented by asterisks (*), * P

Techniques Used: Inhibition, Staining

38) Product Images from "Caveolae and caveolin-1 mediate endocytosis and transcytosis of oxidized low density lipoprotein in endothelial cells"

Article Title: Caveolae and caveolin-1 mediate endocytosis and transcytosis of oxidized low density lipoprotein in endothelial cells

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2010.87

Effects of caveolae-specific inhibitors on DiI-ox-LDL uptake by HUVECs. HUVECs were incubated with DiI-ox-LDL (40 μg/mL) for 30 min at 4 °C. After binding, medium was removed, and cells were incubated at 37 °C for 4 h in the absence or presence of the different caveolae inhibitors. Then HUVECs were washed, fixed, and photographed with an inverted fluorescence microscope (magnification×400). (A) HUVEC were incubated with ox-LDL, (B) HUVEC incubated with DiI-ox-LDL, (C) HUVEC were incubated with DiI-ox-LDL for 30 min at 4 °C, then incubated with carrageenan (250 μg/mL) for 4 h, (D) HUVEC were incubated with DiI-ox-LDL for 30 min at 4 °C, then incubated with filipin (5 μg/mL) for 4 h, (E) HUVEC were incubated with DiI-ox-LDL for 30 min at 4 °C, then incubated with nocodazole (33 μmol/L) for 4 h.
Figure Legend Snippet: Effects of caveolae-specific inhibitors on DiI-ox-LDL uptake by HUVECs. HUVECs were incubated with DiI-ox-LDL (40 μg/mL) for 30 min at 4 °C. After binding, medium was removed, and cells were incubated at 37 °C for 4 h in the absence or presence of the different caveolae inhibitors. Then HUVECs were washed, fixed, and photographed with an inverted fluorescence microscope (magnification×400). (A) HUVEC were incubated with ox-LDL, (B) HUVEC incubated with DiI-ox-LDL, (C) HUVEC were incubated with DiI-ox-LDL for 30 min at 4 °C, then incubated with carrageenan (250 μg/mL) for 4 h, (D) HUVEC were incubated with DiI-ox-LDL for 30 min at 4 °C, then incubated with filipin (5 μg/mL) for 4 h, (E) HUVEC were incubated with DiI-ox-LDL for 30 min at 4 °C, then incubated with nocodazole (33 μmol/L) for 4 h.

Techniques Used: Incubation, Binding Assay, Fluorescence, Microscopy

39) Product Images from "Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments"

Article Title: Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200602080

VEGFR-2 is internalized through a clathrin-dependent pathway. (A) VEC-null and -positive cells were transfected with either Stealth siRNA targeting the mouse clathrin heavy chain or negative Stealth siRNA control duplex (and used 72 h later), or the cells were treated with hypertonic medium (0.45 M sucrose for 30 min) or 1 μg/ml filipin for 1 h. The micrographs show VEGFR-2 immunofluorescence staining after VEGF treatment for 10 min. Both clathrin heavy chain siRNA and hypertonic medium strongly reduced VEGFR-2 vesicular patterning both in VEC-null and -positive cells. Using filipin to interfere with the caveolar compartment had no effect on either cell type. The negative Stealth siRNA control duplex produced results that were indistinguishable from untreated cells (not depicted). Nuclei appear blue after DAPI staining. Bar, 20 μm. (B) Column graphs represent VEGFR-2 vesicular labeling in VEGF-treated cells quantified by ImageJ. Ctr, control; HM, hypertonic medium. Values normalized per cell are the mean of three independent experiments ± SD (error bars). In each experiment, at least five independent fields were analyzed for each point. *, P ≤ 0.01 versus control values by analysis of variance and the Dunnet test.
Figure Legend Snippet: VEGFR-2 is internalized through a clathrin-dependent pathway. (A) VEC-null and -positive cells were transfected with either Stealth siRNA targeting the mouse clathrin heavy chain or negative Stealth siRNA control duplex (and used 72 h later), or the cells were treated with hypertonic medium (0.45 M sucrose for 30 min) or 1 μg/ml filipin for 1 h. The micrographs show VEGFR-2 immunofluorescence staining after VEGF treatment for 10 min. Both clathrin heavy chain siRNA and hypertonic medium strongly reduced VEGFR-2 vesicular patterning both in VEC-null and -positive cells. Using filipin to interfere with the caveolar compartment had no effect on either cell type. The negative Stealth siRNA control duplex produced results that were indistinguishable from untreated cells (not depicted). Nuclei appear blue after DAPI staining. Bar, 20 μm. (B) Column graphs represent VEGFR-2 vesicular labeling in VEGF-treated cells quantified by ImageJ. Ctr, control; HM, hypertonic medium. Values normalized per cell are the mean of three independent experiments ± SD (error bars). In each experiment, at least five independent fields were analyzed for each point. *, P ≤ 0.01 versus control values by analysis of variance and the Dunnet test.

Techniques Used: Transfection, Immunofluorescence, Staining, Produced, Labeling

40) Product Images from "Stigmasterol prevents glucolipotoxicity induced defects in glucose-stimulated insulin secretion"

Article Title: Stigmasterol prevents glucolipotoxicity induced defects in glucose-stimulated insulin secretion

Journal: Scientific Reports

doi: 10.1038/s41598-017-10209-0

Stigmasterol treatment inhibits HGP-induced increase in free cholesterol and ROS. ( a , b ) Free cholesterol but not esterified cholesterol was reduced by stigmasterol. INS-1 cells treated for 72 h with LG, HGP, or HGP + stigmasterol (Stig) were stained with filipin ( a ) and lipidtox ( b ). Scale bars, 10 μm. * p
Figure Legend Snippet: Stigmasterol treatment inhibits HGP-induced increase in free cholesterol and ROS. ( a , b ) Free cholesterol but not esterified cholesterol was reduced by stigmasterol. INS-1 cells treated for 72 h with LG, HGP, or HGP + stigmasterol (Stig) were stained with filipin ( a ) and lipidtox ( b ). Scale bars, 10 μm. * p

Techniques Used: Staining

Glucolipotoxicity increases ROS in β-cells. ( a , b ) Timecourse for HGP-induced ROS production ( a ) and cholesterol accumulation ( b ). INS-1 cells were incubated for up to 48 h with HGP and imaged using carboxy-H 2 DFFDA ( a ) and filipin ( b ), along with Hoechst stain for cell counting. Scale bars, 10 μm. ( c,d ) Background-corrected fluorescence was measured for carboxy-H 2 DFFDA ( c ) and filipin ( d ) fluorescence. * p
Figure Legend Snippet: Glucolipotoxicity increases ROS in β-cells. ( a , b ) Timecourse for HGP-induced ROS production ( a ) and cholesterol accumulation ( b ). INS-1 cells were incubated for up to 48 h with HGP and imaged using carboxy-H 2 DFFDA ( a ) and filipin ( b ), along with Hoechst stain for cell counting. Scale bars, 10 μm. ( c,d ) Background-corrected fluorescence was measured for carboxy-H 2 DFFDA ( c ) and filipin ( d ) fluorescence. * p

Techniques Used: Incubation, Staining, Cell Counting, Fluorescence

ROS induces cholesterol accumulation. ( a ) INS-1 cells were treated with AAPH in concentrations from 0 to 5 mg/ml for 4 h in serum-free media. Free cholesterol was measured using filipin. * p
Figure Legend Snippet: ROS induces cholesterol accumulation. ( a ) INS-1 cells were treated with AAPH in concentrations from 0 to 5 mg/ml for 4 h in serum-free media. Free cholesterol was measured using filipin. * p

Techniques Used:

Glucolipotoxicity increases free cholesterol and decreases insulin secretion. ( a – c ) HGP increases free cholesterol. INS-1 cells ( a ) or human islets ( b ) were treated for 72 h with control growth medium (LG), or HGP. Filipin per cell, indicating free cholesterol, was quantified from 5 independent experiments ( c ). DIC, differential interference contrast. AUI, arbitrary unit of intensity. Scale bars, 10 μm. ( d ) Mevinolin (Mev), an inhibitor of cholesterol synthesis, blocks HGP-induced increase in filipin staining in a dose-dependent manner. Cells were treated with HGP and Mev (μM) in LPDS growth medium for 72 h. ( e ) LPDS or HDL treatment blocks HGP-induced increase in filipin staining. Cells were treated with LG, HGP, HGP in LPDS medium (HGP + LPDS) or HGP and HDL in LPDS medium (HGP + HDL) for 72 h. ( f ) HGP decreases GSIS. Static GSIS assays of INS-1 and human islets were performed in cells treated for 72 h with HGP. ( g – i ) Mev treatment normalizes GSIS ( g ) by inhibiting HGP-induced basal insulin secretion ( h ) without affecting stimulated insulin secretion ( i ). All values are expressed as fold change relative to LG. n = 5 experiments. * p
Figure Legend Snippet: Glucolipotoxicity increases free cholesterol and decreases insulin secretion. ( a – c ) HGP increases free cholesterol. INS-1 cells ( a ) or human islets ( b ) were treated for 72 h with control growth medium (LG), or HGP. Filipin per cell, indicating free cholesterol, was quantified from 5 independent experiments ( c ). DIC, differential interference contrast. AUI, arbitrary unit of intensity. Scale bars, 10 μm. ( d ) Mevinolin (Mev), an inhibitor of cholesterol synthesis, blocks HGP-induced increase in filipin staining in a dose-dependent manner. Cells were treated with HGP and Mev (μM) in LPDS growth medium for 72 h. ( e ) LPDS or HDL treatment blocks HGP-induced increase in filipin staining. Cells were treated with LG, HGP, HGP in LPDS medium (HGP + LPDS) or HGP and HDL in LPDS medium (HGP + HDL) for 72 h. ( f ) HGP decreases GSIS. Static GSIS assays of INS-1 and human islets were performed in cells treated for 72 h with HGP. ( g – i ) Mev treatment normalizes GSIS ( g ) by inhibiting HGP-induced basal insulin secretion ( h ) without affecting stimulated insulin secretion ( i ). All values are expressed as fold change relative to LG. n = 5 experiments. * p

Techniques Used: Staining

41) Product Images from "Gap junction remodeling associated with cholesterol redistribution during fiber cell maturation in the adult chicken lens"

Article Title: Gap junction remodeling associated with cholesterol redistribution during fiber cell maturation in the adult chicken lens

Journal: Molecular Vision

doi:

Filipin-treated gap junctions. A : A representative cholesterol-rich gap junction with the presence of numerous filipin-cholesterol complexes (FCCs, small arrows) is frequently found in the outer cortical fibers. B and C : Cholesterol-intermediate gap junctions with less number of FCCs (small arrows) are observed in the inner cortical fibers. FCCs (small arrows) are often distributed along patches or parallel rows of crystalline-packed connexons (large arrows). D and E : Cholesterol-free gap junctions are mostly seen in the deeper mature cortical fibers. Note that crystalline-arranged connexons can be visualized more clearly on the E-face (ef) of the membrane. In the images, pf indicates the P-face of the membrane. The scale bars indicate 200 nm.
Figure Legend Snippet: Filipin-treated gap junctions. A : A representative cholesterol-rich gap junction with the presence of numerous filipin-cholesterol complexes (FCCs, small arrows) is frequently found in the outer cortical fibers. B and C : Cholesterol-intermediate gap junctions with less number of FCCs (small arrows) are observed in the inner cortical fibers. FCCs (small arrows) are often distributed along patches or parallel rows of crystalline-packed connexons (large arrows). D and E : Cholesterol-free gap junctions are mostly seen in the deeper mature cortical fibers. Note that crystalline-arranged connexons can be visualized more clearly on the E-face (ef) of the membrane. In the images, pf indicates the P-face of the membrane. The scale bars indicate 200 nm.

Techniques Used:

42) Product Images from "'Clustering' SIRP? into the Plasma Membrane Lipid Microdomains Is Required for Activated Monocytes and Macrophages to Mediate Effective Cell Surface Interactions with CD47 "

Article Title: 'Clustering' SIRP? into the Plasma Membrane Lipid Microdomains Is Required for Activated Monocytes and Macrophages to Mediate Effective Cell Surface Interactions with CD47

Journal: PLoS ONE

doi: 10.1371/journal.pone.0077615

Clustering of SIRPα on cell surfaces requires cholesterol-rich lipid rafts. PMA-stimulated THP-1 cells were treated with lipid raft/cholesterol perturbation/modification agents, including MβCD (10 mg/ml), triparanol (5 µM), cholesterol oxidase (CO) (1 unit/ml) and filipin (0.5 µg/ml), prior to A) assessment of SIRPα on the cell surface by immunofluorescence staining, B) analysis of SIRPα localization in lipid rafts by co-staining for raft marker GM1 by cholera toxin B subunit (CT-B), C) assay of SIRPα-mediated cell adhesion to immobilized CD47-AP. **, P
Figure Legend Snippet: Clustering of SIRPα on cell surfaces requires cholesterol-rich lipid rafts. PMA-stimulated THP-1 cells were treated with lipid raft/cholesterol perturbation/modification agents, including MβCD (10 mg/ml), triparanol (5 µM), cholesterol oxidase (CO) (1 unit/ml) and filipin (0.5 µg/ml), prior to A) assessment of SIRPα on the cell surface by immunofluorescence staining, B) analysis of SIRPα localization in lipid rafts by co-staining for raft marker GM1 by cholera toxin B subunit (CT-B), C) assay of SIRPα-mediated cell adhesion to immobilized CD47-AP. **, P

Techniques Used: Modification, Immunofluorescence, Staining, Marker

43) Product Images from "Normalization of Cholesterol Homeostasis by 2-Hydroxypropyl-\u03b2-cyclodextrin in Neurons and Glia from Niemann-Pick C1 (NPC1)-deficient Mice"

Article Title: Normalization of Cholesterol Homeostasis by 2-Hydroxypropyl-\u03b2-cyclodextrin in Neurons and Glia from Niemann-Pick C1 (NPC1)-deficient Mice

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.326405

CD reduces cholesterol sequestration in Npc1 −/− neurons and glia. Filipin staining of unesterified cholesterol in 7-day-old Npc1 −/− cerebellar granule neurons ( A–C ), astrocytes ( D–F ), and microglia ( G–I
Figure Legend Snippet: CD reduces cholesterol sequestration in Npc1 −/− neurons and glia. Filipin staining of unesterified cholesterol in 7-day-old Npc1 −/− cerebellar granule neurons ( A–C ), astrocytes ( D–F ), and microglia ( G–I

Techniques Used: Staining

CD does not alter cholesterol distribution in Npc1 +/+ neurons or astrocytes. Filipin staining of Npc1 +/+ neurons ( A–C ) and astrocytes ( D–F ) incubated for 24 h with vehicle ( Veh ) or indicated CD concentration. Shown are representative images
Figure Legend Snippet: CD does not alter cholesterol distribution in Npc1 +/+ neurons or astrocytes. Filipin staining of Npc1 +/+ neurons ( A–C ) and astrocytes ( D–F ) incubated for 24 h with vehicle ( Veh ) or indicated CD concentration. Shown are representative images

Techniques Used: Staining, Incubation, Concentration Assay

44) Product Images from "Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol"

Article Title: Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1706070114

Ezetimibe is protective in a zebrafish model of S . Typhi infection. ( A ) Fish infected with S . Typhi prgH had increased survival compared with fish infected with WT S . Typhi ( P = 0.01). The survival curve was carried out for 5 d; fish were checked once each day. ( B ) Zebrafish were scored 24 h post S . Typhi infection as cleared (no bacteria), localized (bacteria only in the swim bladder), disseminated (bacteria found outside the swim bladder), or dead (fish dead due to bacterial burden). The swim bladders are denoted by red circles; bacteria are denoted by the red arrows. ( C ) Fish infected with the S . Typhi prgH mutant had increased clearance of bacteria at 24 h ( P = 0.03). ( D ) Ezetimibe had no effect on S . Typhi bacterial growth. Bacteria were diluted from an overnight stock and grown with DMSO or 10 µM ezetimibe. The OD 600 was taken every 30 min for 3.5 h. Data points are the mean from two separate experiments. ( E ) Ezetimibe decreased filipin staining in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe reduced filipin (0.05 mg/mL) staining ( P = 0.003) in zebrafish; n = 20 fish from two separate experiments; P value from an unpaired t test. ( F ) Fish pretreated with ezetimibe had increased survival from S . Typhi infection compared with DMSO-pretreated controls ( P = 0.03). ( G ) Ezetimibe treatment increased bacterial clearance in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe increased the percentage of fish that cleared the bacteria 24 h postinfection from 8 to 30% ( P = 0.009). Infection data for each survival curve and clearance comparisons are from three independent experiments with a minimum of n = 60 fish. P values from survival curves are from the Mantel–Cox test; P values for other comparisons are from unpaired t tests.
Figure Legend Snippet: Ezetimibe is protective in a zebrafish model of S . Typhi infection. ( A ) Fish infected with S . Typhi prgH had increased survival compared with fish infected with WT S . Typhi ( P = 0.01). The survival curve was carried out for 5 d; fish were checked once each day. ( B ) Zebrafish were scored 24 h post S . Typhi infection as cleared (no bacteria), localized (bacteria only in the swim bladder), disseminated (bacteria found outside the swim bladder), or dead (fish dead due to bacterial burden). The swim bladders are denoted by red circles; bacteria are denoted by the red arrows. ( C ) Fish infected with the S . Typhi prgH mutant had increased clearance of bacteria at 24 h ( P = 0.03). ( D ) Ezetimibe had no effect on S . Typhi bacterial growth. Bacteria were diluted from an overnight stock and grown with DMSO or 10 µM ezetimibe. The OD 600 was taken every 30 min for 3.5 h. Data points are the mean from two separate experiments. ( E ) Ezetimibe decreased filipin staining in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe reduced filipin (0.05 mg/mL) staining ( P = 0.003) in zebrafish; n = 20 fish from two separate experiments; P value from an unpaired t test. ( F ) Fish pretreated with ezetimibe had increased survival from S . Typhi infection compared with DMSO-pretreated controls ( P = 0.03). ( G ) Ezetimibe treatment increased bacterial clearance in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe increased the percentage of fish that cleared the bacteria 24 h postinfection from 8 to 30% ( P = 0.009). Infection data for each survival curve and clearance comparisons are from three independent experiments with a minimum of n = 60 fish. P values from survival curves are from the Mantel–Cox test; P values for other comparisons are from unpaired t tests.

Techniques Used: Infection, Fluorescence In Situ Hybridization, Mutagenesis, Staining

45) Product Images from "cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells"

Article Title: cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

doi: 10.1152/ajplung.90402.2008

Correction of cholesterol transport in CF model IB3 cells with Rp -cAMPS. A : representative image of IB3 cells costained for βarr2 and endogenous free cholesterol (filipin). B : representative images of CF model IB3 cells costained for βarr2
Figure Legend Snippet: Correction of cholesterol transport in CF model IB3 cells with Rp -cAMPS. A : representative image of IB3 cells costained for βarr2 and endogenous free cholesterol (filipin). B : representative images of CF model IB3 cells costained for βarr2

Techniques Used:

Correction of cholesterol transport in CF model cells with Rp -cAMPS. A : representative image of WT 9/HTEo- cells costained for βarr2 and endogenous free cholesterol (filipin). B : 3 representative images of CF model 9/HTEo- pCEPR cells costained
Figure Legend Snippet: Correction of cholesterol transport in CF model cells with Rp -cAMPS. A : representative image of WT 9/HTEo- cells costained for βarr2 and endogenous free cholesterol (filipin). B : 3 representative images of CF model 9/HTEo- pCEPR cells costained

Techniques Used:

Cholesterol accumulation in 9/HTEo- cells initiated by 8-bromo-cAMP (8-Br-cAMP). A : 3 representative images of endogenous free cholesterol content visualized by filipin staining in wild-type (WT) 9/HTEo- (pCEP) cells in the absence or presence of the
Figure Legend Snippet: Cholesterol accumulation in 9/HTEo- cells initiated by 8-bromo-cAMP (8-Br-cAMP). A : 3 representative images of endogenous free cholesterol content visualized by filipin staining in wild-type (WT) 9/HTEo- (pCEP) cells in the absence or presence of the

Techniques Used: Staining

β 2 -Adrenergic receptor (β2-AR) and cholesterol colocalize in CF model 9/HTEo- pCEPR cells. Immunostaining for β2-AR and cholesterol visualization with filipin in WT 9/HTEo- pCEP ( A ) and CF model pCEPR cells ( B ) is shown. In merged
Figure Legend Snippet: β 2 -Adrenergic receptor (β2-AR) and cholesterol colocalize in CF model 9/HTEo- pCEPR cells. Immunostaining for β2-AR and cholesterol visualization with filipin in WT 9/HTEo- pCEP ( A ) and CF model pCEPR cells ( B ) is shown. In merged

Techniques Used: Immunostaining

46) Product Images from "Targeted cytoplasmic irradiation induces bystander responses"

Article Title: Targeted cytoplasmic irradiation induces bystander responses

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0404930101

Cytoplasmic traversal-induced MN production in the T98G population ( A ) and bystander AG0 population ( B ) with or without filipin treatment. One or 10 cells in the T98G population were individually irradiated through the cytoplasm with five helium ions ( * , P
Figure Legend Snippet: Cytoplasmic traversal-induced MN production in the T98G population ( A ) and bystander AG0 population ( B ) with or without filipin treatment. One or 10 cells in the T98G population were individually irradiated through the cytoplasm with five helium ions ( * , P

Techniques Used: Irradiation

Percentage of NO-positive cells ( A ) and relative NO-induced fluorescence intensity ( B ) in the cytoplasmic-traversed T98G population with or without treatment with c-PTIO or filipin. The fluorescence intensity of the nonirradiated T98G was considered as 1. A few cells within the T98G population were individually traversed through cytoplasm with one helium ion ( * , P
Figure Legend Snippet: Percentage of NO-positive cells ( A ) and relative NO-induced fluorescence intensity ( B ) in the cytoplasmic-traversed T98G population with or without treatment with c-PTIO or filipin. The fluorescence intensity of the nonirradiated T98G was considered as 1. A few cells within the T98G population were individually traversed through cytoplasm with one helium ion ( * , P

Techniques Used: Fluorescence

47) Product Images from "Synphilin-1 Enhances ?-Synuclein Aggregation in Yeast and Contributes to Cellular Stress and Cell Death in a Sir2-Dependent Manner"

Article Title: Synphilin-1 Enhances ?-Synuclein Aggregation in Yeast and Contributes to Cellular Stress and Cell Death in a Sir2-Dependent Manner

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013700

Synphilin-1 interaction with lipid rafts in yeast membranes. A: Co-localization of inclusions formed by dsRed-SY WT with lipid particles and endomembranes (Bodipy 500/510; upper panels), lipid droplets (Bodipy 505/515; middle panels), or sterol-enriched membrane domains (Filipin; lower panels) in yeast. B and C: Interaction with lipid rafts of SY WT , SY R621C or α-Syn–EGFP in yeast membranes of the wild-type BY4741 strain (B) or its isogenic erg24Δ mutant (C). The proteins expressed are indicated on the right, the antibodies used for immunodetection on the left. Lipid raft fractions (boxed areas) were identified using the yeast lipid raft marker Pma1. The open arrowhead indicates the position of the 250 kDa molecular weight marker, the black arrowhead that of the 150 kDa marker.
Figure Legend Snippet: Synphilin-1 interaction with lipid rafts in yeast membranes. A: Co-localization of inclusions formed by dsRed-SY WT with lipid particles and endomembranes (Bodipy 500/510; upper panels), lipid droplets (Bodipy 505/515; middle panels), or sterol-enriched membrane domains (Filipin; lower panels) in yeast. B and C: Interaction with lipid rafts of SY WT , SY R621C or α-Syn–EGFP in yeast membranes of the wild-type BY4741 strain (B) or its isogenic erg24Δ mutant (C). The proteins expressed are indicated on the right, the antibodies used for immunodetection on the left. Lipid raft fractions (boxed areas) were identified using the yeast lipid raft marker Pma1. The open arrowhead indicates the position of the 250 kDa molecular weight marker, the black arrowhead that of the 150 kDa marker.

Techniques Used: Mutagenesis, Immunodetection, Marker, Molecular Weight

48) Product Images from "Endolysosome involvement in LDL cholesterol-induced Alzheimer's disease-like pathology in primary cultured neurons"

Article Title: Endolysosome involvement in LDL cholesterol-induced Alzheimer's disease-like pathology in primary cultured neurons

Journal: Life sciences

doi: 10.1016/j.lfs.2012.04.039

LDL cholesterol increased cholesterol accumulation in neurons. (A) Neurons treated with LDL cholesterol (50 µg/ml) for 3 days exhibited altered free cholesterol (filipin staining) distribution; cholesterol was distributed near the plasma membrane
Figure Legend Snippet: LDL cholesterol increased cholesterol accumulation in neurons. (A) Neurons treated with LDL cholesterol (50 µg/ml) for 3 days exhibited altered free cholesterol (filipin staining) distribution; cholesterol was distributed near the plasma membrane

Techniques Used: Staining

49) Product Images from "Selective Roles for Cholesterol and Actin in Compartmentalization of Different Proteins in the Golgi and Plasma Membrane of Polarized Cells *Selective Roles for Cholesterol and Actin in Compartmentalization of Different Proteins in the Golgi and Plasma Membrane of Polarized Cells * S⃞"

Article Title: Selective Roles for Cholesterol and Actin in Compartmentalization of Different Proteins in the Golgi and Plasma Membrane of Polarized Cells *Selective Roles for Cholesterol and Actin in Compartmentalization of Different Proteins in the Golgi and Plasma Membrane of Polarized Cells * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M803819200

Cholesterol plays a major role in the organization of proteins in apical and basolateral domains of polarized MDCK cells. A , apparent diffusion coefficients ( D ) of the four studied proteins at steady state ( colored bars ) and upon depletion of cholesterol synthesis by treatment with mevinolin ( white bars ). B , confocal pictures of filipin staining at apical pole ( panels a and b ) and basolateral pole ( panels c and d ) of polarized MDCK cells in control conditions ( panels a and c ) and upon loading of cholesterol ( panels b and d ). Bar , 10 μm. C , quantification of the filipin staining in polarized MDCK cells loaded with cholesterol. Intensity fluorescence is expressed as percentage of control cells. The experiments were performed three independent times, and the error bars are the mean ± S.D. D , apparent diffusion coefficient ( D ) of proteins of interest at steady state ( colored bars ) and upon addition of cholesterol ( white bars ). Fitted data are shown from at least three independent experiments (for A and D ) with n > 15. The error bars are the means ± S.D. * , p
Figure Legend Snippet: Cholesterol plays a major role in the organization of proteins in apical and basolateral domains of polarized MDCK cells. A , apparent diffusion coefficients ( D ) of the four studied proteins at steady state ( colored bars ) and upon depletion of cholesterol synthesis by treatment with mevinolin ( white bars ). B , confocal pictures of filipin staining at apical pole ( panels a and b ) and basolateral pole ( panels c and d ) of polarized MDCK cells in control conditions ( panels a and c ) and upon loading of cholesterol ( panels b and d ). Bar , 10 μm. C , quantification of the filipin staining in polarized MDCK cells loaded with cholesterol. Intensity fluorescence is expressed as percentage of control cells. The experiments were performed three independent times, and the error bars are the mean ± S.D. D , apparent diffusion coefficient ( D ) of proteins of interest at steady state ( colored bars ) and upon addition of cholesterol ( white bars ). Fitted data are shown from at least three independent experiments (for A and D ) with n > 15. The error bars are the means ± S.D. * , p

Techniques Used: Diffusion-based Assay, Staining, Fluorescence

50) Product Images from "Loss of Niemann Pick type C proteins 1 and 2 greatly enhances HIV infectivity and is associated with accumulation of HIV Gag and cholesterol in late endosomes/lysosomes"

Article Title: Loss of Niemann Pick type C proteins 1 and 2 greatly enhances HIV infectivity and is associated with accumulation of HIV Gag and cholesterol in late endosomes/lysosomes

Journal: Virology Journal

doi: 10.1186/1743-422X-9-31

Gag localizes to LE/L compartments in NPC-deficient cells . At 96 h post-infection with HIV-1, fibroblasts were stained and analyzed by fluorescence microscopy to assess Gag localization. Intracellular cholesterol (blue) was visualized by staining the cells with filipin dye ( A, E, I, M ). LysoTracker was used to stain late endosomes/lysosomes (red) ( C, G, K, O ) and HIV-1 Gag protein (green) ( B, F, J, N ) was stained with KC57-FITC.
Figure Legend Snippet: Gag localizes to LE/L compartments in NPC-deficient cells . At 96 h post-infection with HIV-1, fibroblasts were stained and analyzed by fluorescence microscopy to assess Gag localization. Intracellular cholesterol (blue) was visualized by staining the cells with filipin dye ( A, E, I, M ). LysoTracker was used to stain late endosomes/lysosomes (red) ( C, G, K, O ) and HIV-1 Gag protein (green) ( B, F, J, N ) was stained with KC57-FITC.

Techniques Used: Infection, Staining, Fluorescence, Microscopy

51) Product Images from "The Role of the Niemann-Pick Disease, Type C1 Protein in Adipocyte Insulin Action"

Article Title: The Role of the Niemann-Pick Disease, Type C1 Protein in Adipocyte Insulin Action

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095598

Pharmacological and genetic inhibition of NPC1 reduced insulin signalling in 3T3-L1 adipocytes. NPC1 was inhibited in 3T3-L1 adipocytes through treatment with U18666a or through siRNA-mediated knockdown of NPC1. ( a ) Subcellular localisation of cholesterol in 3T3-L1 adipocytes after treatment with U18666a for indicated times was visualised by Filipin staining (scale bar = 10 µm), bottom panel. Corresponding bright field images are provided (top panel). Images are representative of n ≥300 cells viewed per condition over 3 experiments. ( b ) Total membrane cholesterol levels within the total membrane fraction. Data are mean ± S.E.M, n = 3 independent experiments, one-way ANOVA. ( c ) Insulin signalling was assessed by immunoblotting total cell lysates of unstimulated and insulin-stimulated (100 nM, 20 min) 3T3-L1 adipocytes for phospho-T308 Akt, phospho-S473 Akt, phospho-T642 AS160 and 14-3-3. ( d ) Total protein expression of key proteins in the insulin pathway determined by immunoblotting for IRS-1, IRβ and Akt. ( e ) Subcellular localisation of cholesterol after treatment with scrambled or NPC1 siRNA was visualised by Filipin staining (scale bar = 10 µm). Images are representative of n≥300 cells viewed per condition over 3 experiments. ( f ) The extent of siRNA mediated knockdown of NPC1 and the effect of knockdown on expression of proximal insulin signalling components was measured by probing for NPC1, IRS-1, IRβ and Akt. ( g ) The effect of NPC1 knock-down on insulin signalling was assessed by immunoblotting with phospho-T308 Akt, phospho-S473 Akt, phospho-T642 AS160 and 14-3-3 antibodies. ( h ) The levels of insulin-stimulated pS473 and pT308 of Akt in cells NPC1 knockdown cells was quantified relative to control cells. Data are mean ± S.E.M, n = 3 independent experiments, two-sample t-test. ( i ) 3T3-L1 adipocytes overexpressing PDGF receptor were treated with U18666a for 72 hr prior to assessment of insulin and PDGF-mediated (20 ng/mL, 20 min) signalling. Signalling was assessed by immunoblotting with phospho-T308 Akt, phospho-S473 Akt and 14-3-3 antibodies. All data are representative of at least n = 3 independent experiments, significance calculated compared to control cells, n.s = non-significant, *, = p
Figure Legend Snippet: Pharmacological and genetic inhibition of NPC1 reduced insulin signalling in 3T3-L1 adipocytes. NPC1 was inhibited in 3T3-L1 adipocytes through treatment with U18666a or through siRNA-mediated knockdown of NPC1. ( a ) Subcellular localisation of cholesterol in 3T3-L1 adipocytes after treatment with U18666a for indicated times was visualised by Filipin staining (scale bar = 10 µm), bottom panel. Corresponding bright field images are provided (top panel). Images are representative of n ≥300 cells viewed per condition over 3 experiments. ( b ) Total membrane cholesterol levels within the total membrane fraction. Data are mean ± S.E.M, n = 3 independent experiments, one-way ANOVA. ( c ) Insulin signalling was assessed by immunoblotting total cell lysates of unstimulated and insulin-stimulated (100 nM, 20 min) 3T3-L1 adipocytes for phospho-T308 Akt, phospho-S473 Akt, phospho-T642 AS160 and 14-3-3. ( d ) Total protein expression of key proteins in the insulin pathway determined by immunoblotting for IRS-1, IRβ and Akt. ( e ) Subcellular localisation of cholesterol after treatment with scrambled or NPC1 siRNA was visualised by Filipin staining (scale bar = 10 µm). Images are representative of n≥300 cells viewed per condition over 3 experiments. ( f ) The extent of siRNA mediated knockdown of NPC1 and the effect of knockdown on expression of proximal insulin signalling components was measured by probing for NPC1, IRS-1, IRβ and Akt. ( g ) The effect of NPC1 knock-down on insulin signalling was assessed by immunoblotting with phospho-T308 Akt, phospho-S473 Akt, phospho-T642 AS160 and 14-3-3 antibodies. ( h ) The levels of insulin-stimulated pS473 and pT308 of Akt in cells NPC1 knockdown cells was quantified relative to control cells. Data are mean ± S.E.M, n = 3 independent experiments, two-sample t-test. ( i ) 3T3-L1 adipocytes overexpressing PDGF receptor were treated with U18666a for 72 hr prior to assessment of insulin and PDGF-mediated (20 ng/mL, 20 min) signalling. Signalling was assessed by immunoblotting with phospho-T308 Akt, phospho-S473 Akt and 14-3-3 antibodies. All data are representative of at least n = 3 independent experiments, significance calculated compared to control cells, n.s = non-significant, *, = p

Techniques Used: Inhibition, Staining, Expressing

52) Product Images from "Cellular uptake mechanism and intracellular fate of hydrophobically modified pullulan nanoparticles"

Article Title: Cellular uptake mechanism and intracellular fate of hydrophobically modified pullulan nanoparticles

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S44342

Effect of endocytic inhibitor on the internalization of FITC-CHSP NPs. Notes: Hepg2 cells were pretreated with chlorpromazine (7 μg/mL), filipin (1 μg/mL), or amiloride (50 μM), in serum-free medium for 1 hour, and then treated with FITC-CHSP NPs (0.15 mg/mL, 2 hours, 37°C). The group in the presence of FITC-CHSP NPs but without inhibitor treatment was used as control, and their uptake was expressed as 100%. * P
Figure Legend Snippet: Effect of endocytic inhibitor on the internalization of FITC-CHSP NPs. Notes: Hepg2 cells were pretreated with chlorpromazine (7 μg/mL), filipin (1 μg/mL), or amiloride (50 μM), in serum-free medium for 1 hour, and then treated with FITC-CHSP NPs (0.15 mg/mL, 2 hours, 37°C). The group in the presence of FITC-CHSP NPs but without inhibitor treatment was used as control, and their uptake was expressed as 100%. * P

Techniques Used:

53) Product Images from "Entry of Francisella tularensis into Murine B Cells: The Role of B Cell Receptors and Complement Receptors"

Article Title: Entry of Francisella tularensis into Murine B Cells: The Role of B Cell Receptors and Complement Receptors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0132571

Disturbance of lipid rafts. For disturbing lipid rafts, the cholesterol-binding agent filipin or methyl-beta cyclodextrin (Cyclodex) was used. The peritoneal B cells were pretreated with 10 μg/mL filipin or 10 mM cyclodextrin and consequently infected with (A) F . tularensis LVS/GFP or (B) opsonized F . tularensis LVS/GFP with complement. Entry into all B cells (CD19 + ) and individual B cell subsets was detected by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t -test was used to test for significant differences between untreated B cells and cyclodextrin- or filipin-treated cells (*** P
Figure Legend Snippet: Disturbance of lipid rafts. For disturbing lipid rafts, the cholesterol-binding agent filipin or methyl-beta cyclodextrin (Cyclodex) was used. The peritoneal B cells were pretreated with 10 μg/mL filipin or 10 mM cyclodextrin and consequently infected with (A) F . tularensis LVS/GFP or (B) opsonized F . tularensis LVS/GFP with complement. Entry into all B cells (CD19 + ) and individual B cell subsets was detected by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed t -test was used to test for significant differences between untreated B cells and cyclodextrin- or filipin-treated cells (*** P

Techniques Used: Binding Assay, Infection, Flow Cytometry, Cytometry, Two Tailed Test

54) Product Images from "Integrin α6β4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues"

Article Title: Integrin α6β4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues

Journal: Cell Death and Differentiation

doi: 10.1038/s41418-018-0114-7

Integrin β4 and α6 dimerize during the IR-induced cellular senescence. a–c A549 cells pre-incubated with an inhibitor of integrin β4 (ASC-8) or Src (PP2) were irradiated with 6 Gy IR. SA-β-Gal positivity was observed 3 days after irradiation. Scale bars indicate 10 μm ( a ). Immunoblotting was performed with the indicated antibodies at various time intervals after irradiation. Actin served as the loading control ( b , c ). d A549 cells pre-incubated with 0.5 mM MβCD were irradiated with 6 Gy IR. At 1 h post-irradiation, immunoprecipitates were prepared with anti-α-integrin β4, and then immunoblotted with anti-α-integrin α6. e A549 cells were irradiated with 6 Gy and incubated for 1 h, and lipid rafts were fractionated. Equal volumes of lipid rafts (Rafts; fractions 8–10) and nonlipid rafts (Nonrafts; fractions 3–6) were resolved by SDS-PAGE, and immunoblotting was performed with the indicated antibodies. Actin was used as a marker of nonraft fractions. f A549 cells were irradiated with 6 Gy, incubated for the indicated times, and then labeled with filipin and Alexa Fluor 488 phalloidin. The fluorescence intensities of filipin per area (mm 2 ) were quantified from 15 different cells using the ImageJ program. Scale bars indicate 20 μm . g A549 cells were irradiated with 6 Gy and incubated for the indicated times, and the cholesterol contents in cells were measured using an Amplex Red cholesterol assay kit. The results are expressed with respect to the amount of protein. h , i A549 cells pre-incubated with oleic acid were irradiated with 6 Gy IR. Cell lysates prepared at the indicated time intervals were immunoblotted ( h ), and SA-β-Gal staining ( i ) was performed. Scale bars indicate 10 μm. The values represent the mean ± SD of three independent experiments; *** indicates p
Figure Legend Snippet: Integrin β4 and α6 dimerize during the IR-induced cellular senescence. a–c A549 cells pre-incubated with an inhibitor of integrin β4 (ASC-8) or Src (PP2) were irradiated with 6 Gy IR. SA-β-Gal positivity was observed 3 days after irradiation. Scale bars indicate 10 μm ( a ). Immunoblotting was performed with the indicated antibodies at various time intervals after irradiation. Actin served as the loading control ( b , c ). d A549 cells pre-incubated with 0.5 mM MβCD were irradiated with 6 Gy IR. At 1 h post-irradiation, immunoprecipitates were prepared with anti-α-integrin β4, and then immunoblotted with anti-α-integrin α6. e A549 cells were irradiated with 6 Gy and incubated for 1 h, and lipid rafts were fractionated. Equal volumes of lipid rafts (Rafts; fractions 8–10) and nonlipid rafts (Nonrafts; fractions 3–6) were resolved by SDS-PAGE, and immunoblotting was performed with the indicated antibodies. Actin was used as a marker of nonraft fractions. f A549 cells were irradiated with 6 Gy, incubated for the indicated times, and then labeled with filipin and Alexa Fluor 488 phalloidin. The fluorescence intensities of filipin per area (mm 2 ) were quantified from 15 different cells using the ImageJ program. Scale bars indicate 20 μm . g A549 cells were irradiated with 6 Gy and incubated for the indicated times, and the cholesterol contents in cells were measured using an Amplex Red cholesterol assay kit. The results are expressed with respect to the amount of protein. h , i A549 cells pre-incubated with oleic acid were irradiated with 6 Gy IR. Cell lysates prepared at the indicated time intervals were immunoblotted ( h ), and SA-β-Gal staining ( i ) was performed. Scale bars indicate 10 μm. The values represent the mean ± SD of three independent experiments; *** indicates p

Techniques Used: Incubation, Irradiation, SDS Page, Marker, Labeling, Fluorescence, Amplex Red Cholesterol Assay, Staining

55) Product Images from "Degradation of aggregated LDL occurs in complex extracellular sub-compartments of the lysosomal synapse"

Article Title: Degradation of aggregated LDL occurs in complex extracellular sub-compartments of the lysosomal synapse

Journal: Journal of Cell Science

doi: 10.1242/jcs.181743

AgLDL is sequestered in compartments that are surface-connected and contain acidified sub-regions where free cholesterol is generated. (A–D) Ratiometric confocal images of J774 cells incubated with agLDL dual-labeled with a pH-sensitive and pH-insensitive probe for 1 h. (A) Aggregate contained above the surface of the cells and (B) agLDL in the middle of the cells. (C) Enlargement of an area containing acidified regions, highlighted by a box in B. Regions of acidification that represent completely internalized vesicles (arrowheads) and areas of low pH that retain their connection to portions of the aggregate that are at neutral pH (arrows) can be seen. (D) 3D reconstruction of the data shown in A and B. (E–H) RAW264.7 macrophage-like cells were incubated with Alexa633–agLDL (red) for 1 h, stained with Alexa555–CtB (blue) on ice to label the plasma membrane, fixed and stained with filipin (green) to visualize free cholesterol. (E) A cell is contacting Alexa633–agLDL. Some filipin labeling of free cholesterol can be seen inside the cell (arrowheads). (F–H) An extracellular strand of agLDL in close proximity to the cells is shown by an arrow in the inset (F). Single color images of the same region show that it is labeled with filipin (G), but the cell only touches this strand at the base (H). There are areas of filipin labeling that are also labeled with CtB, suggesting that these are also associated with the plasma membrane.
Figure Legend Snippet: AgLDL is sequestered in compartments that are surface-connected and contain acidified sub-regions where free cholesterol is generated. (A–D) Ratiometric confocal images of J774 cells incubated with agLDL dual-labeled with a pH-sensitive and pH-insensitive probe for 1 h. (A) Aggregate contained above the surface of the cells and (B) agLDL in the middle of the cells. (C) Enlargement of an area containing acidified regions, highlighted by a box in B. Regions of acidification that represent completely internalized vesicles (arrowheads) and areas of low pH that retain their connection to portions of the aggregate that are at neutral pH (arrows) can be seen. (D) 3D reconstruction of the data shown in A and B. (E–H) RAW264.7 macrophage-like cells were incubated with Alexa633–agLDL (red) for 1 h, stained with Alexa555–CtB (blue) on ice to label the plasma membrane, fixed and stained with filipin (green) to visualize free cholesterol. (E) A cell is contacting Alexa633–agLDL. Some filipin labeling of free cholesterol can be seen inside the cell (arrowheads). (F–H) An extracellular strand of agLDL in close proximity to the cells is shown by an arrow in the inset (F). Single color images of the same region show that it is labeled with filipin (G), but the cell only touches this strand at the base (H). There are areas of filipin labeling that are also labeled with CtB, suggesting that these are also associated with the plasma membrane.

Techniques Used: Generated, Incubation, Labeling, Staining, CtB Assay

56) Product Images from "Acid sphingomyelinase involvement in tumor necrosis factor ?-regulated vascular and steroid disruption during luteolysis in vivo"

Article Title: Acid sphingomyelinase involvement in tumor necrosis factor ?-regulated vascular and steroid disruption during luteolysis in vivo

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0712260105

TNF induces apoptosis of mouse CL MVECs. ( a ) Treatment with 50 ng/ml TNF induced apoptosis of isolated MVECs, which was prevented by treatment with 15 μg/ml ETA. ( b ) Treatment with 50 ng/ml TNF or an inactive ceramide dihydro ceramide (50 μM DHC) did not induce apoptosis of mouse luteinized granulosa cells, whereas treatment with active ceramide analogues (50 μM C2 and 200 nM C16) or 100 milliunits/ml rASMase increased apoptosis. ( c ) Treatment with 0.5 μg/ml filipin to block ceramide accumulation inhibited TNF-induced apoptosis of MVECs.
Figure Legend Snippet: TNF induces apoptosis of mouse CL MVECs. ( a ) Treatment with 50 ng/ml TNF induced apoptosis of isolated MVECs, which was prevented by treatment with 15 μg/ml ETA. ( b ) Treatment with 50 ng/ml TNF or an inactive ceramide dihydro ceramide (50 μM DHC) did not induce apoptosis of mouse luteinized granulosa cells, whereas treatment with active ceramide analogues (50 μM C2 and 200 nM C16) or 100 milliunits/ml rASMase increased apoptosis. ( c ) Treatment with 0.5 μg/ml filipin to block ceramide accumulation inhibited TNF-induced apoptosis of MVECs.

Techniques Used: Isolation, Blocking Assay

57) Product Images from "Dual Roles for RHOA/RHO-Kinase In the Regulated Trafficking of a Voltage-sensitive Potassium Channel"

Article Title: Dual Roles for RHOA/RHO-Kinase In the Regulated Trafficking of a Voltage-sensitive Potassium Channel

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E08-10-1074

Steady-state Kv1.2 homeostasis is composed of constitutive cholesterol-dependent endocytosis and of constitutive recycling. (A) Application of the sterol-binding agent filipin (7.7 μM; 1 h) to HEK-K cells significantly increases steady-state surface Kv1.2 as detected by flow cytometry. Subsequent treatment with Y27632 (10 μM; 30 min) does not elicit a further increase (n = 32; *p
Figure Legend Snippet: Steady-state Kv1.2 homeostasis is composed of constitutive cholesterol-dependent endocytosis and of constitutive recycling. (A) Application of the sterol-binding agent filipin (7.7 μM; 1 h) to HEK-K cells significantly increases steady-state surface Kv1.2 as detected by flow cytometry. Subsequent treatment with Y27632 (10 μM; 30 min) does not elicit a further increase (n = 32; *p

Techniques Used: Binding Assay, Flow Cytometry, Cytometry

58) Product Images from "Diabetes Adversely Affects Macrophages During Atherosclerotic Plaque Regression in Mice"

Article Title: Diabetes Adversely Affects Macrophages During Atherosclerotic Plaque Regression in Mice

Journal: Diabetes

doi: 10.2337/db10-0778

Effects of diabetes on the lipid content of aortic root sections after plasma lipid reduction. Aortic roots were sectioned, fixed, and stained for neutral lipids (presumably cholesteryl esters) using Oil-Red O or for free cholesterol using filipin. The stained areas were quantified by Image Pro Plus software. Oil-Red O–positive areas ( A ) and free cholesterol (blue staining) ( B ) are quantified and presented as the percentage of total plaque area. Sample pictures of each lipid stain are shown for all experimental groups in C and D . In addition to filipin staining, immunostaining for CD68 (red) and β-actin (green) are also shown ( D ). Data (means ± SEM) were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison test. P
Figure Legend Snippet: Effects of diabetes on the lipid content of aortic root sections after plasma lipid reduction. Aortic roots were sectioned, fixed, and stained for neutral lipids (presumably cholesteryl esters) using Oil-Red O or for free cholesterol using filipin. The stained areas were quantified by Image Pro Plus software. Oil-Red O–positive areas ( A ) and free cholesterol (blue staining) ( B ) are quantified and presented as the percentage of total plaque area. Sample pictures of each lipid stain are shown for all experimental groups in C and D . In addition to filipin staining, immunostaining for CD68 (red) and β-actin (green) are also shown ( D ). Data (means ± SEM) were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison test. P

Techniques Used: Staining, Software, Immunostaining

59) Product Images from "Clinically used selective oestrogen receptor modulators increase LDL receptor activity in primary human lymphocytes"

Article Title: Clinically used selective oestrogen receptor modulators increase LDL receptor activity in primary human lymphocytes

Journal: British Journal of Pharmacology

doi: 10.1111/bph.13016

Effects of tamoxifen, raloxifene and toremifene on cholesterol distribution in lymphocytes. Lymphocytes from male donors were treated with DiI-LDL and vehicle (control) or 5 μM SERMs for 24 h. Then, cells were fixed, stained with filipin and examined for filipin and DiI fluorescence. Photographs correspond to lymphocytes from one subject representative of three. Bar, 10 μm.
Figure Legend Snippet: Effects of tamoxifen, raloxifene and toremifene on cholesterol distribution in lymphocytes. Lymphocytes from male donors were treated with DiI-LDL and vehicle (control) or 5 μM SERMs for 24 h. Then, cells were fixed, stained with filipin and examined for filipin and DiI fluorescence. Photographs correspond to lymphocytes from one subject representative of three. Bar, 10 μm.

Techniques Used: Staining, Fluorescence

60) Product Images from "ABCA1 Protein Enhances Toll-like Receptor 4 (TLR4)-stimulated Interleukin-10 (IL-10) Secretion through Protein Kinase A (PKA) Activation *"

Article Title: ABCA1 Protein Enhances Toll-like Receptor 4 (TLR4)-stimulated Interleukin-10 (IL-10) Secretion through Protein Kinase A (PKA) Activation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.413245

Cholesterol depletion increases p-CREB. A–C , RAW macrophages were treated with 0, 1, or 5 m m MCD for 30 min ( A ), 3 μg/ml filipin for 1 h ( B ), or 3, 5, or 7 μ m statins (compactin and simvastatin) for 48 h ( C ). Cell lysates were
Figure Legend Snippet: Cholesterol depletion increases p-CREB. A–C , RAW macrophages were treated with 0, 1, or 5 m m MCD for 30 min ( A ), 3 μg/ml filipin for 1 h ( B ), or 3, 5, or 7 μ m statins (compactin and simvastatin) for 48 h ( C ). Cell lysates were

Techniques Used:

Cholesterol depletion promotes IL-10 secretion but suppresses TNF-α release. A , RAW macrophages were treated with 10 μ m T0901317 overnight and followed by 100 ng/ml LPS treatment in combination with MCD or filipin for 6 h. B , some of the
Figure Legend Snippet: Cholesterol depletion promotes IL-10 secretion but suppresses TNF-α release. A , RAW macrophages were treated with 10 μ m T0901317 overnight and followed by 100 ng/ml LPS treatment in combination with MCD or filipin for 6 h. B , some of the

Techniques Used:

Cholesterol depletion increases PKA-phosphorylated proteins. A and B , RAW macrophages were pretreated with or without 50 μ m PKI for 1 h and subsequently treated 30 min with 5 m m MCD or 10 μg/ml filipin ( A ) or incubated with statins in
Figure Legend Snippet: Cholesterol depletion increases PKA-phosphorylated proteins. A and B , RAW macrophages were pretreated with or without 50 μ m PKI for 1 h and subsequently treated 30 min with 5 m m MCD or 10 μg/ml filipin ( A ) or incubated with statins in

Techniques Used: Incubation

61) Product Images from "Reproductive Pathology and Sperm Physiology in Acid Sphingomyelinase-Deficient Mice"

Article Title: Reproductive Pathology and Sperm Physiology in Acid Sphingomyelinase-Deficient Mice

Journal: The American Journal of Pathology

doi:

Cholesterol accumulation in caudal spermatozoa at 6 months of age. A: Representative freeze-fracture PF-face images of filipin-sterol complexes, FSC (original magnifications, ×10,000; insets : ×60,000). B: Surface analysis of FSC. The steepness of the spectral slope is indicative of image surface coarseness. C: In situ plasma membrane cholesterol efflux.
Figure Legend Snippet: Cholesterol accumulation in caudal spermatozoa at 6 months of age. A: Representative freeze-fracture PF-face images of filipin-sterol complexes, FSC (original magnifications, ×10,000; insets : ×60,000). B: Surface analysis of FSC. The steepness of the spectral slope is indicative of image surface coarseness. C: In situ plasma membrane cholesterol efflux.

Techniques Used: In Situ

62) Product Images from "A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells"

Article Title: A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells

Journal: Molecular Cancer

doi: 10.1186/1476-4598-10-69

Vacuoles induced by MIPP are derived from macropinosomes that undergo progressive fusion events and accumulate at a pre-lysosomal stage . A) Time-lapse phase-contrast microscopy of U251 cells treated with MIPP. Images were captured at 30 sec intervals during the period between 13-80 min after the addition of 10 μM MIPP, and the images were assembled into a movie, which is available as Movie 1 (Additional file 1 ). The panels show sequential snapshots from the movie, with the elapsed time after addition of MIPP (min) indicated. Newly formed macropinosomes can be seen fusing with each other to form larger vacuoles. The small two-headed arrows point to vesicles that have fused in the subsequent frame. B) U251 cells were treated with 10 μM MIPP for 4 h, then incubated with the indicated fluorescent tracer or organelle marker, as described in Materials and Methods. The same field of cells is depicted in the matching phase-contrast and fluorescent images. In the top panel, the arrows indicate some of the specific vacuoles that have incorporated the Lucifer yellow. C) Cells were pretreated for 30 min with 12 μg/ml filipin or an equivalent volume of vehicle (DMSO), then 10 μM MIPP was added to the cultures. Phase-contrast images were acquired 100 min after the addition of the MIPP. D) Bafilomycin A1 (Baf-A) blocks the induction of vacuoles by MIPP. Cells were pretreated for 1 h in the presence (+) or absence (-) of 100 nM Baf A prior to addition of MIPP (+MIPP) or DMSO (-MIPP). Phase-contrast images were taken 1 h after addition of MIPP. The scale bars in all of the images represent 10 microns.
Figure Legend Snippet: Vacuoles induced by MIPP are derived from macropinosomes that undergo progressive fusion events and accumulate at a pre-lysosomal stage . A) Time-lapse phase-contrast microscopy of U251 cells treated with MIPP. Images were captured at 30 sec intervals during the period between 13-80 min after the addition of 10 μM MIPP, and the images were assembled into a movie, which is available as Movie 1 (Additional file 1 ). The panels show sequential snapshots from the movie, with the elapsed time after addition of MIPP (min) indicated. Newly formed macropinosomes can be seen fusing with each other to form larger vacuoles. The small two-headed arrows point to vesicles that have fused in the subsequent frame. B) U251 cells were treated with 10 μM MIPP for 4 h, then incubated with the indicated fluorescent tracer or organelle marker, as described in Materials and Methods. The same field of cells is depicted in the matching phase-contrast and fluorescent images. In the top panel, the arrows indicate some of the specific vacuoles that have incorporated the Lucifer yellow. C) Cells were pretreated for 30 min with 12 μg/ml filipin or an equivalent volume of vehicle (DMSO), then 10 μM MIPP was added to the cultures. Phase-contrast images were acquired 100 min after the addition of the MIPP. D) Bafilomycin A1 (Baf-A) blocks the induction of vacuoles by MIPP. Cells were pretreated for 1 h in the presence (+) or absence (-) of 100 nM Baf A prior to addition of MIPP (+MIPP) or DMSO (-MIPP). Phase-contrast images were taken 1 h after addition of MIPP. The scale bars in all of the images represent 10 microns.

Techniques Used: Derivative Assay, Microscopy, Size-exclusion Chromatography, Incubation, Marker

63) Product Images from "A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking"

Article Title: A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201004142

Rescue effects of ORP5 expression. (A) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with EGFP alone or RNAi-resistant GFP-OPR5 (GFP-ORP5-siR) for 6 h. Cells then received medium D supplemented with 50 µg/ml LDL for 18 h followed by processing for filipin staining and fluorescence microscopy. Images are shown to indicate that cholesterol accumulation was significantly alleviated in GFP-ORP5-siR–expressing cells (71 ± 2%) but not in GFP-expressing cells (5 ± 3%). (B) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with RNAi-resistant GFP-OPR5 for 24 h. Cells were fixed and labeled with antibodies to TGN46. Confocal images are shown to indicate that the dispersed distribution of TGN46 was rescued in cells expressing GFP-ORP5. (C) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with RNAi-resistant mCherry-OPR5 for 24 h. Cells were fixed and labeled with antibodies to CI-MPR. Confocal images are shown to indicate that the dispersed distribution of CI-MPR was rescued in cells expressing mCherry-ORP5. Bars, 10 µm.
Figure Legend Snippet: Rescue effects of ORP5 expression. (A) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with EGFP alone or RNAi-resistant GFP-OPR5 (GFP-ORP5-siR) for 6 h. Cells then received medium D supplemented with 50 µg/ml LDL for 18 h followed by processing for filipin staining and fluorescence microscopy. Images are shown to indicate that cholesterol accumulation was significantly alleviated in GFP-ORP5-siR–expressing cells (71 ± 2%) but not in GFP-expressing cells (5 ± 3%). (B) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with RNAi-resistant GFP-OPR5 for 24 h. Cells were fixed and labeled with antibodies to TGN46. Confocal images are shown to indicate that the dispersed distribution of TGN46 was rescued in cells expressing GFP-ORP5. (C) HeLa cells grown in medium A were transfected with siORP5 for 48 h, followed by transfection with RNAi-resistant mCherry-OPR5 for 24 h. Cells were fixed and labeled with antibodies to CI-MPR. Confocal images are shown to indicate that the dispersed distribution of CI-MPR was rescued in cells expressing mCherry-ORP5. Bars, 10 µm.

Techniques Used: Expressing, Transfection, Staining, Fluorescence, Microscopy, Labeling

The localization of DsRed-Golgi in cells depleted for ORP5, NPC1, or both. (A–D) HeLa cells grown in medium A were transfected with siCTRL (A), siNPC1 (B), siORP5 (C), or siNPC1 and siORP5 (D) for 48 h, followed by transfection with the Golgi marker DsRed-Golgi for 6 h. Cells were treated with 50 µg/ml LDL in medium D for 18 h followed by processing for filipin staining. Confocal fluorescence images are shown. Data are representative of three independent experiments with similar results. Enlarged views of the boxed regions are shown in the insets. Bars, 10 µm. (E) Quantification of DsRed-Golgi mislocalization in A–D. Values are the percentage of transfected cells with mislocalized DsRed-Golgi marker (means + SD [error bars], n > 80; **, P
Figure Legend Snippet: The localization of DsRed-Golgi in cells depleted for ORP5, NPC1, or both. (A–D) HeLa cells grown in medium A were transfected with siCTRL (A), siNPC1 (B), siORP5 (C), or siNPC1 and siORP5 (D) for 48 h, followed by transfection with the Golgi marker DsRed-Golgi for 6 h. Cells were treated with 50 µg/ml LDL in medium D for 18 h followed by processing for filipin staining. Confocal fluorescence images are shown. Data are representative of three independent experiments with similar results. Enlarged views of the boxed regions are shown in the insets. Bars, 10 µm. (E) Quantification of DsRed-Golgi mislocalization in A–D. Values are the percentage of transfected cells with mislocalized DsRed-Golgi marker (means + SD [error bars], n > 80; **, P

Techniques Used: Transfection, Marker, Staining, Fluorescence

ORP5 knockdown causes endosomal cholesterol accumulation and inhibits LDL-stimulated cholesterol esterification. (A) HeLa cells grown in medium A were transfected with siNPC1, siORP5, siORP8, or a universal control siCTRL for 48 h, followed by transfection with pcDNA4-ORP5 or pmCherry-ORP8 for 24 h. Efficiency of knockdown was analyzed by immunoblotting using polyclonal anti-NPC1, anti-ORP5, or anti-DsRed antibodies. The molecular mass of the proteins is indicated in kilodaltons. (B) HeLa cells grown in medium A were transfected with the indicated siRNAs for 54 h. Cells then received medium D supplemented with 50 µg/ml LDL for 18 h followed by processing for filipin staining. Fluorescent images are representative of four independent experiments with similar results. Bars, 10 µm. (C) Cells that received the same treatments as in B were lysed by 0.1 M NaOH and processed for measuring cellular free cholesterol as described in Materials and methods. Values were normalized to total cell proteins and are relative to siCTRL-transfected cells. The results are expressed as means + SD (error bars). **, P
Figure Legend Snippet: ORP5 knockdown causes endosomal cholesterol accumulation and inhibits LDL-stimulated cholesterol esterification. (A) HeLa cells grown in medium A were transfected with siNPC1, siORP5, siORP8, or a universal control siCTRL for 48 h, followed by transfection with pcDNA4-ORP5 or pmCherry-ORP8 for 24 h. Efficiency of knockdown was analyzed by immunoblotting using polyclonal anti-NPC1, anti-ORP5, or anti-DsRed antibodies. The molecular mass of the proteins is indicated in kilodaltons. (B) HeLa cells grown in medium A were transfected with the indicated siRNAs for 54 h. Cells then received medium D supplemented with 50 µg/ml LDL for 18 h followed by processing for filipin staining. Fluorescent images are representative of four independent experiments with similar results. Bars, 10 µm. (C) Cells that received the same treatments as in B were lysed by 0.1 M NaOH and processed for measuring cellular free cholesterol as described in Materials and methods. Values were normalized to total cell proteins and are relative to siCTRL-transfected cells. The results are expressed as means + SD (error bars). **, P

Techniques Used: Transfection, Staining

Cholesterol accumulates on the limiting membrane of LE/LY in ORP5 knockdown cells. (A) HeLa cells grown in medium A were transfected with siNPC1, siORP5, siNPC1, and siORP5, or a universal control siCTRL for 48 h, followed by transfection with pcDNA4-ORP5 for 24 h. Efficiency of knockdown was analyzed by immunoblotting using polyclonal anti-NPC1, anti-ORP5 antibodies. The molecular mass of the proteins is indicated in kilodaltons next to the gel blot. (B) HeLa cells grown in medium A were transfected with the indicated siRNAs for 48 h, followed by incubation in medium D for 18 h. Cells were then chased with 50 µg/ml LDL and [ 14 C]-palmitate in medium D for 6 h. A cholesterol esterification assay was performed as described in Fig. 3 D , and relative cholesterol esterification was quantified as described in Fig. 3 E . The results are expressed as means + SD (error bars). (C) HeLa cells grown in medium A were transfected with indicated siRNAs for 54 h. Cells then received medium D supplemented with 50 µg/ml LDL for 18 h followed by processing for filipin staining. Fluorescent images are representative of four independent experiments with similar results. Bars, 10 µm. (D and E) HeLa cells grown in medium A were transfected with siORP5 (D) or siNPC1 (E) for 48 h, followed by transfection with pEYFP-Lamp1 for 6 h. Cells then received medium D supplemented with 50 µg/ml LDL for 18 h followed by processing for filipin staining and fluorescence microscopy. Enlarged views of the boxed regions are shown in the bottom panels. Arrows indicate the overlapping of accumulated cholesterol with EYFP-Lamp-1 in ORP5 knockdown cells but not in NPC1 knockdown cells. Bars, 10 µm.
Figure Legend Snippet: Cholesterol accumulates on the limiting membrane of LE/LY in ORP5 knockdown cells. (A) HeLa cells grown in medium A were transfected with siNPC1, siORP5, siNPC1, and siORP5, or a universal control siCTRL for 48 h, followed by transfection with pcDNA4-ORP5 for 24 h. Efficiency of knockdown was analyzed by immunoblotting using polyclonal anti-NPC1, anti-ORP5 antibodies. The molecular mass of the proteins is indicated in kilodaltons next to the gel blot. (B) HeLa cells grown in medium A were transfected with the indicated siRNAs for 48 h, followed by incubation in medium D for 18 h. Cells were then chased with 50 µg/ml LDL and [ 14 C]-palmitate in medium D for 6 h. A cholesterol esterification assay was performed as described in Fig. 3 D , and relative cholesterol esterification was quantified as described in Fig. 3 E . The results are expressed as means + SD (error bars). (C) HeLa cells grown in medium A were transfected with indicated siRNAs for 54 h. Cells then received medium D supplemented with 50 µg/ml LDL for 18 h followed by processing for filipin staining. Fluorescent images are representative of four independent experiments with similar results. Bars, 10 µm. (D and E) HeLa cells grown in medium A were transfected with siORP5 (D) or siNPC1 (E) for 48 h, followed by transfection with pEYFP-Lamp1 for 6 h. Cells then received medium D supplemented with 50 µg/ml LDL for 18 h followed by processing for filipin staining and fluorescence microscopy. Enlarged views of the boxed regions are shown in the bottom panels. Arrows indicate the overlapping of accumulated cholesterol with EYFP-Lamp-1 in ORP5 knockdown cells but not in NPC1 knockdown cells. Bars, 10 µm.

Techniques Used: Transfection, Western Blot, Incubation, Staining, Fluorescence, Microscopy

64) Product Images from "Visualization of DC-SIGN-Mediated Entry Pathway of Engineered Lentiviral Vectors in Target Cells"

Article Title: Visualization of DC-SIGN-Mediated Entry Pathway of Engineered Lentiviral Vectors in Target Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0067400

DC-SIGN-mediated and clathrin-dependent entry of LV-SVGmu. (A) Viruses binding to DC-SIGN-expressing cells. 293T/hDC-SIGN cells were incubated with GFP-Vpr-labeled LV-SVGmu particles (green) for 30 min at 4°C, fixed, and immunostained with anti-DC-SIGN antibody (red). (B) Real-time monitoring of virus internalization mediated by the DC-SIGN receptor. 293T cells seeded on a glass-bottom dish were transiently transfected with DsRed-DC-SIGN (red). At 24 h post-transfection, the cells were incubated with GFP-Vpr-labeled LV-SVGmu particles (green) for 30 min at 4°C and were then warmed to 37°C to initiate virus internalization. Confocal time-lapse images were then recorded. Selected frames of the real-time imaging are presented. (C) 293T/hDC-SIGN cells were incubated with GFP-Vpr-labeled LV-SVGmu viruses (green) for 30 min at 4°C to synchronize infection and were then shifted to 37°C for 5 min. After incubation, cells were fixed, permeabilized and immunostained with antibody against caveolin-1 (red) or clathrin (red). The boxed regions are enlarged in the bottom panels. Scale bar represents 5 µm. (D) Quantification of GFP-Vpr-labeled LV-SVGmu particles colocalized with clathrin signals. Overlap coefficents were calculated using Mander’s overlap coefficient in Nikon NIS-Elements software by viewing more than 40 cells. (E) The effect of inhibitory drugs on LV-SVGmu or LV-SFV transduction. 293T/hDC-SIGN cells were preincubated with chlorpromazine (CPZ, 30 nM) or filipin (15 nM) for 30 min at 37°C. Cells were then infected with LV-SVGmu or LV-SFV encoding a reporter GFP gene for 90 min in the presence of drugs. After an additional 3 h of incubation, the medium was replaced with fresh media, and the percentage of GFP-positive cells was analyzed by flow cytometry after 72 h. All of the data were then normalized based on the actual percentage of GFP-positive cells of the no drug treatment group of LV-SVGmu (35.2±0.028%) or LV-SFV (33.5±0.34%). All data are shown as the means of triplicate experiments. Asterisk indicates comparison to the no drug treatment group (* P
Figure Legend Snippet: DC-SIGN-mediated and clathrin-dependent entry of LV-SVGmu. (A) Viruses binding to DC-SIGN-expressing cells. 293T/hDC-SIGN cells were incubated with GFP-Vpr-labeled LV-SVGmu particles (green) for 30 min at 4°C, fixed, and immunostained with anti-DC-SIGN antibody (red). (B) Real-time monitoring of virus internalization mediated by the DC-SIGN receptor. 293T cells seeded on a glass-bottom dish were transiently transfected with DsRed-DC-SIGN (red). At 24 h post-transfection, the cells were incubated with GFP-Vpr-labeled LV-SVGmu particles (green) for 30 min at 4°C and were then warmed to 37°C to initiate virus internalization. Confocal time-lapse images were then recorded. Selected frames of the real-time imaging are presented. (C) 293T/hDC-SIGN cells were incubated with GFP-Vpr-labeled LV-SVGmu viruses (green) for 30 min at 4°C to synchronize infection and were then shifted to 37°C for 5 min. After incubation, cells were fixed, permeabilized and immunostained with antibody against caveolin-1 (red) or clathrin (red). The boxed regions are enlarged in the bottom panels. Scale bar represents 5 µm. (D) Quantification of GFP-Vpr-labeled LV-SVGmu particles colocalized with clathrin signals. Overlap coefficents were calculated using Mander’s overlap coefficient in Nikon NIS-Elements software by viewing more than 40 cells. (E) The effect of inhibitory drugs on LV-SVGmu or LV-SFV transduction. 293T/hDC-SIGN cells were preincubated with chlorpromazine (CPZ, 30 nM) or filipin (15 nM) for 30 min at 37°C. Cells were then infected with LV-SVGmu or LV-SFV encoding a reporter GFP gene for 90 min in the presence of drugs. After an additional 3 h of incubation, the medium was replaced with fresh media, and the percentage of GFP-positive cells was analyzed by flow cytometry after 72 h. All of the data were then normalized based on the actual percentage of GFP-positive cells of the no drug treatment group of LV-SVGmu (35.2±0.028%) or LV-SFV (33.5±0.34%). All data are shown as the means of triplicate experiments. Asterisk indicates comparison to the no drug treatment group (* P

Techniques Used: Binding Assay, Expressing, Incubation, Labeling, Transfection, Imaging, Infection, Software, Transduction, Flow Cytometry, Cytometry

65) Product Images from "Impaired Cholesterol Biosynthesis in a Neuronal Cell Line Persistently Infected with Measles Virus ▿Impaired Cholesterol Biosynthesis in a Neuronal Cell Line Persistently Infected with Measles Virus ▿ †"

Article Title: Impaired Cholesterol Biosynthesis in a Neuronal Cell Line Persistently Infected with Measles Virus ▿Impaired Cholesterol Biosynthesis in a Neuronal Cell Line Persistently Infected with Measles Virus ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.01880-08

Decreased cellular content of cholesterol in NS20Y/MS cells. NS20Y and NS20Y/MS cells were probed for cellular cholesterol using filipin staining and analyzed by flow cytometry as described in Materials and Methods. (A) A histogram overlay representing
Figure Legend Snippet: Decreased cellular content of cholesterol in NS20Y/MS cells. NS20Y and NS20Y/MS cells were probed for cellular cholesterol using filipin staining and analyzed by flow cytometry as described in Materials and Methods. (A) A histogram overlay representing

Techniques Used: Mass Spectrometry, Staining, Flow Cytometry, Cytometry

66) Product Images from "Role of the Brucella suis Lipopolysaccharide O Antigen in Phagosomal Genesis and in Inhibition of Phagosome-Lysosome Fusion in Murine Macrophages "

Article Title: Role of the Brucella suis Lipopolysaccharide O Antigen in Phagosomal Genesis and in Inhibition of Phagosome-Lysosome Fusion in Murine Macrophages

Journal: Infection and Immunity

doi: 10.1128/IAI.71.3.1481-1490.2003

Effect of filipin, β-methyl cyclodextrin, and cholera toxin B subunit on short-term survival of nonopsonized and opsonized B. suis and B. suis manB ( manB mutant) inside murine macrophages. Cells were pretreated with cholesterol-binding (filipin, 8 μg/ml) or -depleting (β-methyl cyclodextrin, 10 mM) molecules or the ganglioside GM1-binding molecule (cholera toxin B subunit, 40 μg/ml). Following treatment with the relevant drug, infection with Brucella spp., and postinfection incubation for 1 h, cells were lysed in Triton X-100 and intracellular viability at this short time (1 h) was measured as described in Materials and Methods. For each strain or condition, the control (100% value) was obtained in the absence of drug treatment. Experiments were performed in triplicate, and the values represent means ± standard deviations. The experiments were repeated at least four times.
Figure Legend Snippet: Effect of filipin, β-methyl cyclodextrin, and cholera toxin B subunit on short-term survival of nonopsonized and opsonized B. suis and B. suis manB ( manB mutant) inside murine macrophages. Cells were pretreated with cholesterol-binding (filipin, 8 μg/ml) or -depleting (β-methyl cyclodextrin, 10 mM) molecules or the ganglioside GM1-binding molecule (cholera toxin B subunit, 40 μg/ml). Following treatment with the relevant drug, infection with Brucella spp., and postinfection incubation for 1 h, cells were lysed in Triton X-100 and intracellular viability at this short time (1 h) was measured as described in Materials and Methods. For each strain or condition, the control (100% value) was obtained in the absence of drug treatment. Experiments were performed in triplicate, and the values represent means ± standard deviations. The experiments were repeated at least four times.

Techniques Used: Mutagenesis, Binding Assay, Infection, Incubation

67) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Utilizes an Actin Polymerization-Dependent Macropinocytic Pathway To Enter Human Dermal Microvascular Endothelial and Human Umbilical Vein Endothelial Cells ▿"

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Utilizes an Actin Polymerization-Dependent Macropinocytic Pathway To Enter Human Dermal Microvascular Endothelial and Human Umbilical Vein Endothelial Cells ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02498-08

Effect of endocytic inhibitors on KSHV gene expression, binding, and entry in HMVEC-d and HFF cells. (A and B) Cells were left untreated or pretreated with endocytic inhibitors for 1 h at 37°C, washed, and infected with KSHV at an MOI of 10. Total RNA was isolated at 2 h and 24 h p.i., and 50 ng of DNase-treated RNA/μl was subjected to real-time RT-PCR with ORF73 and ORF50 gene-specific primers and TaqMan probes. Known concentrations of DNase-treated, in vitro-transcribed ORF50 and ORF73 transcripts were used in real-time RT-PCR to construct a standard graph from which the relative copy numbers of viral transcripts were calculated and normalized to the amount of GAPDH. Histograms depict KSHV ORF73 and ORF50 gene RNA copy numbers in untreated cells (KSHV) or cells in the presence of indicated nontoxic concentrations of chlorpromazine (Chlor), EIPA, rottlerin (Rot), cytochalasin D (CytoD), filipin, cholera toxin B (CTB), MβCD, or nystatin (nys) in HMVEC-d (A) and HFF (B) cells. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three independent experiments. (C, D, and E) Effect of endocytic inhibitors on KSHV binding (C) and internalization (D) in HMVEC-d cells and on internalization (E) in HFF cells. (C) HMVEC-d cells grown in 24-well plates were either left untreated or pretreated with various nontoxic concentrations of agents for 1 h at 37°C and incubated with a fixed concentration of [ 3 ). Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments.
Figure Legend Snippet: Effect of endocytic inhibitors on KSHV gene expression, binding, and entry in HMVEC-d and HFF cells. (A and B) Cells were left untreated or pretreated with endocytic inhibitors for 1 h at 37°C, washed, and infected with KSHV at an MOI of 10. Total RNA was isolated at 2 h and 24 h p.i., and 50 ng of DNase-treated RNA/μl was subjected to real-time RT-PCR with ORF73 and ORF50 gene-specific primers and TaqMan probes. Known concentrations of DNase-treated, in vitro-transcribed ORF50 and ORF73 transcripts were used in real-time RT-PCR to construct a standard graph from which the relative copy numbers of viral transcripts were calculated and normalized to the amount of GAPDH. Histograms depict KSHV ORF73 and ORF50 gene RNA copy numbers in untreated cells (KSHV) or cells in the presence of indicated nontoxic concentrations of chlorpromazine (Chlor), EIPA, rottlerin (Rot), cytochalasin D (CytoD), filipin, cholera toxin B (CTB), MβCD, or nystatin (nys) in HMVEC-d (A) and HFF (B) cells. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three independent experiments. (C, D, and E) Effect of endocytic inhibitors on KSHV binding (C) and internalization (D) in HMVEC-d cells and on internalization (E) in HFF cells. (C) HMVEC-d cells grown in 24-well plates were either left untreated or pretreated with various nontoxic concentrations of agents for 1 h at 37°C and incubated with a fixed concentration of [ 3 ). Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments.

Techniques Used: Expressing, Binding Assay, Infection, Isolation, Quantitative RT-PCR, In Vitro, Construct, CtB Assay, Standard Deviation, Incubation, Concentration Assay

68) Product Images from "Hexokinase II–derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells"

Article Title: Hexokinase II–derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells

Journal: The FASEB Journal

doi: 10.1096/fj.201601173R

Determination of cellular uptake mechanisms of pHK and pHK-PAS. A ) Effects of low temperature and energy depletion on peptide internalization. HeLa cells were preincubated for 1 h at 4°C in serum-free DMEM or pretreated for 1 h at 37°C with 10 mM sodium azide and 6 mM 2-deoxy- d -glucose in serum- and glucose-free DMEM to deplete cellular ATP. pHK A488 (25 µM; left) or pHK-PAS A488 (25 µM; middle) was then added, and cells were maintained for 2 h at 4°C or in the presence of sodium azide/2-deoxy- d -glucose at 37°C. B ) Effects of endocytosis inhibitors on peptide internalization. HeLa cells were treated for 30 min at 37°C in serum-free DMEM with the following: 10 µM chlorpromazine (Chlor; clathrin-dependent endocytosis), 5 mM methyl-β-cyclodextrin (MβCD; lipid raft–mediated endocytosis), 4 µM filipin (Filip; caveolae-dependent endocytosis), 10 µM nocodazole (Nocod; microtubule polymerization), or 10 µM cytochalasin D (Cyto D; macropinocytosis). Cells were then treated with 25 µM pHK A488 (left) or pHK-PAS A488 (middle) and maintained for 2 h at 37°C in the presence of inhibitors and peptides. Thereafter, cells were washed 3 times with ice-cold PBS, trypsinized, centrifuged, and resuspended in ice-cold PBS with 10% FBS, and fluorescence was measured by FACS. Cells that were treated with peptide without inhibitors at 37°C were used as control, and cells that were treated with vehicle alone served as background. Uptake efficiencies (right) were determined from the ratio of fluorescence of cells treated with peptide under different inhibition conditions to control cells. ns, nonsignificant ( P > 0.05). ** P
Figure Legend Snippet: Determination of cellular uptake mechanisms of pHK and pHK-PAS. A ) Effects of low temperature and energy depletion on peptide internalization. HeLa cells were preincubated for 1 h at 4°C in serum-free DMEM or pretreated for 1 h at 37°C with 10 mM sodium azide and 6 mM 2-deoxy- d -glucose in serum- and glucose-free DMEM to deplete cellular ATP. pHK A488 (25 µM; left) or pHK-PAS A488 (25 µM; middle) was then added, and cells were maintained for 2 h at 4°C or in the presence of sodium azide/2-deoxy- d -glucose at 37°C. B ) Effects of endocytosis inhibitors on peptide internalization. HeLa cells were treated for 30 min at 37°C in serum-free DMEM with the following: 10 µM chlorpromazine (Chlor; clathrin-dependent endocytosis), 5 mM methyl-β-cyclodextrin (MβCD; lipid raft–mediated endocytosis), 4 µM filipin (Filip; caveolae-dependent endocytosis), 10 µM nocodazole (Nocod; microtubule polymerization), or 10 µM cytochalasin D (Cyto D; macropinocytosis). Cells were then treated with 25 µM pHK A488 (left) or pHK-PAS A488 (middle) and maintained for 2 h at 37°C in the presence of inhibitors and peptides. Thereafter, cells were washed 3 times with ice-cold PBS, trypsinized, centrifuged, and resuspended in ice-cold PBS with 10% FBS, and fluorescence was measured by FACS. Cells that were treated with peptide without inhibitors at 37°C were used as control, and cells that were treated with vehicle alone served as background. Uptake efficiencies (right) were determined from the ratio of fluorescence of cells treated with peptide under different inhibition conditions to control cells. ns, nonsignificant ( P > 0.05). ** P

Techniques Used: Fluorescence, FACS, Inhibition

69) Product Images from "Targeting cholesterol-rich microdomains to circumvent tamoxifen-resistant breast cancer"

Article Title: Targeting cholesterol-rich microdomains to circumvent tamoxifen-resistant breast cancer

Journal: Breast Cancer Research : BCR

doi: 10.1186/bcr3063

Cholesterol-rich lipid microdomains are involved in α-TEA + TAM circumvention of TAMR . (a) MCF-7/TAMR and MCF-7/HER-2 cells were treated with α-TEA at 20 μ M for 15 hours. Identification of cholesterol-rich microdomains was determined by staining the cells with fluorescein-tagged cholesterol marker Filipin and images viewed by using a fluorescence microscope. (b) MCF-7/TAMR cells were pretreated with 10 μ M exogenous cholesterol for 2 hours, followed by treatment with 20 μ M α-TEA or 20 μ M α-TEA plus 1 μ M TAM for l day. Western blot analyses were performed to determine treatment effects on prosurvival signaling mediators and PARP cleavage. (a, b) Data are representative of three independent experiments. α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue; MCF-7/HER-2, clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; PARP, poly (ADP-ribose) polymerase; TAM, tamoxifen; TAMR, tamoxifen resistant.
Figure Legend Snippet: Cholesterol-rich lipid microdomains are involved in α-TEA + TAM circumvention of TAMR . (a) MCF-7/TAMR and MCF-7/HER-2 cells were treated with α-TEA at 20 μ M for 15 hours. Identification of cholesterol-rich microdomains was determined by staining the cells with fluorescein-tagged cholesterol marker Filipin and images viewed by using a fluorescence microscope. (b) MCF-7/TAMR cells were pretreated with 10 μ M exogenous cholesterol for 2 hours, followed by treatment with 20 μ M α-TEA or 20 μ M α-TEA plus 1 μ M TAM for l day. Western blot analyses were performed to determine treatment effects on prosurvival signaling mediators and PARP cleavage. (a, b) Data are representative of three independent experiments. α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue; MCF-7/HER-2, clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; PARP, poly (ADP-ribose) polymerase; TAM, tamoxifen; TAMR, tamoxifen resistant.

Techniques Used: Staining, Marker, Fluorescence, Microscopy, Western Blot

TAMR cells in comparison to TAMS cells constitutively express higher levels of prosurvival mediators and cholesterol-rich lipid microdomains . (a) TAMR cell lines MCF-7/TAMR and MCF-7/HER-2, as well as TAMS parental MCF-7/TAMS and MCF-7/Neo cells, were treated with TAM at 1 μ M or VEH (ethanol) in steroid-depleted media containing 17β-estradiol (10 -9 M ) for 2 days. Molecular profile of prosurvival mediators was determined with Western blot analyses. (pHER-1/tHER-1 and pHER-2/tHER-2 were not detected in the MCF-7/TAMS and MCF-7/Neo cells). (b) The expression of lipid microdomains was determined by staining the cells with fluorescein-tagged DiLC16 lipid-raft marker and viewed with a fluorescence microscope. (c) The expression of cholesterol-rich lipid microdomains was determined by staining cells with fluorescein-labeled filipin, a cholesterol marker, and viewed by using a fluorescence microscope. (a-c) Data are representative of a minimum of three independent experiments. DiIC-16, dialkylindocarbocyanine; HER, human epidermal growth factor; MCF-7/HER-2, Clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; MCF-7/TAMS, TAM-sensitive MCF-7/parental; pHER-1, phosphorylated-HER-1; pHER-2, phosphorylated-HER-2; TAM, tamoxifen; TAMR, tamoxifen resistant; TAMS, tamoxifen sensitive; VEH, vehicle control.
Figure Legend Snippet: TAMR cells in comparison to TAMS cells constitutively express higher levels of prosurvival mediators and cholesterol-rich lipid microdomains . (a) TAMR cell lines MCF-7/TAMR and MCF-7/HER-2, as well as TAMS parental MCF-7/TAMS and MCF-7/Neo cells, were treated with TAM at 1 μ M or VEH (ethanol) in steroid-depleted media containing 17β-estradiol (10 -9 M ) for 2 days. Molecular profile of prosurvival mediators was determined with Western blot analyses. (pHER-1/tHER-1 and pHER-2/tHER-2 were not detected in the MCF-7/TAMS and MCF-7/Neo cells). (b) The expression of lipid microdomains was determined by staining the cells with fluorescein-tagged DiLC16 lipid-raft marker and viewed with a fluorescence microscope. (c) The expression of cholesterol-rich lipid microdomains was determined by staining cells with fluorescein-labeled filipin, a cholesterol marker, and viewed by using a fluorescence microscope. (a-c) Data are representative of a minimum of three independent experiments. DiIC-16, dialkylindocarbocyanine; HER, human epidermal growth factor; MCF-7/HER-2, Clone 18 MCF-7 cells overexpressing HER-2; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; MCF-7/TAMS, TAM-sensitive MCF-7/parental; pHER-1, phosphorylated-HER-1; pHER-2, phosphorylated-HER-2; TAM, tamoxifen; TAMR, tamoxifen resistant; TAMS, tamoxifen sensitive; VEH, vehicle control.

Techniques Used: Western Blot, Expressing, Staining, Marker, Fluorescence, Microscopy, Labeling

70) Product Images from "Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments"

Article Title: Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200602080

VEGFR-2 is internalized through a clathrin-dependent pathway. (A) VEC-null and -positive cells were transfected with either Stealth siRNA targeting the mouse clathrin heavy chain or negative Stealth siRNA control duplex (and used 72 h later), or the cells were treated with hypertonic medium (0.45 M sucrose for 30 min) or 1 μg/ml filipin for 1 h. The micrographs show VEGFR-2 immunofluorescence staining after VEGF treatment for 10 min. Both clathrin heavy chain siRNA and hypertonic medium strongly reduced VEGFR-2 vesicular patterning both in VEC-null and -positive cells. Using filipin to interfere with the caveolar compartment had no effect on either cell type. The negative Stealth siRNA control duplex produced results that were indistinguishable from untreated cells (not depicted). Nuclei appear blue after DAPI staining. Bar, 20 μm. (B) Column graphs represent VEGFR-2 vesicular labeling in VEGF-treated cells quantified by ImageJ. Ctr, control; HM, hypertonic medium. Values normalized per cell are the mean of three independent experiments ± SD (error bars). In each experiment, at least five independent fields were analyzed for each point. *, P ≤ 0.01 versus control values by analysis of variance and the Dunnet test.
Figure Legend Snippet: VEGFR-2 is internalized through a clathrin-dependent pathway. (A) VEC-null and -positive cells were transfected with either Stealth siRNA targeting the mouse clathrin heavy chain or negative Stealth siRNA control duplex (and used 72 h later), or the cells were treated with hypertonic medium (0.45 M sucrose for 30 min) or 1 μg/ml filipin for 1 h. The micrographs show VEGFR-2 immunofluorescence staining after VEGF treatment for 10 min. Both clathrin heavy chain siRNA and hypertonic medium strongly reduced VEGFR-2 vesicular patterning both in VEC-null and -positive cells. Using filipin to interfere with the caveolar compartment had no effect on either cell type. The negative Stealth siRNA control duplex produced results that were indistinguishable from untreated cells (not depicted). Nuclei appear blue after DAPI staining. Bar, 20 μm. (B) Column graphs represent VEGFR-2 vesicular labeling in VEGF-treated cells quantified by ImageJ. Ctr, control; HM, hypertonic medium. Values normalized per cell are the mean of three independent experiments ± SD (error bars). In each experiment, at least five independent fields were analyzed for each point. *, P ≤ 0.01 versus control values by analysis of variance and the Dunnet test.

Techniques Used: Transfection, Immunofluorescence, Staining, Produced, Labeling

71) Product Images from "Essential role of membrane cholesterol in accelerated BCR internalization and uncoupling from NF-?B in B cell clonal anergy"

Article Title: Essential role of membrane cholesterol in accelerated BCR internalization and uncoupling from NF-?B in B cell clonal anergy

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20060552

Effects of depleting cholesterol from membranes of naive and anergic B cells. (A) Surface levels of raft-associated GM1 sphingolipids and IgD a were measured by flow cytometry before (gray histogram) and after treatment with the indicated concentrations of MBCD (open histograms). The dotted lines serve as an arbitrary visual reference for control fluorescence levels. (B) Membrane cholesterol levels were measured by staining with filipin and flow cytometric measurement of filipin fluorescence on untreated cells (Control), MBCD-treated cells (MBCD), or cells treated with MBCD that had been preloaded with cholesterol (MBCD-Chol). (C) Values of the mean fluorescence of the filipin staining were normalized with the value obtained for untreated and unstimulated BCR tg cells. Open bars, BCR tg cells; filled bars, BCR tg x HEL tg cells. Data shown are representative of two to five independent experiments. (D) HEL antigen-induced calcium responses were recorded as variations in the Indo-1 fluorescence ratio gated on the B220 + cell population. Bold lines, calcium response of control cells; thin lines, cells that were first pretreated with 2.5 mM MBCD. Stimulation is performed after 60 s with soluble HEL. Experiment shown is representative of five independent experiments.
Figure Legend Snippet: Effects of depleting cholesterol from membranes of naive and anergic B cells. (A) Surface levels of raft-associated GM1 sphingolipids and IgD a were measured by flow cytometry before (gray histogram) and after treatment with the indicated concentrations of MBCD (open histograms). The dotted lines serve as an arbitrary visual reference for control fluorescence levels. (B) Membrane cholesterol levels were measured by staining with filipin and flow cytometric measurement of filipin fluorescence on untreated cells (Control), MBCD-treated cells (MBCD), or cells treated with MBCD that had been preloaded with cholesterol (MBCD-Chol). (C) Values of the mean fluorescence of the filipin staining were normalized with the value obtained for untreated and unstimulated BCR tg cells. Open bars, BCR tg cells; filled bars, BCR tg x HEL tg cells. Data shown are representative of two to five independent experiments. (D) HEL antigen-induced calcium responses were recorded as variations in the Indo-1 fluorescence ratio gated on the B220 + cell population. Bold lines, calcium response of control cells; thin lines, cells that were first pretreated with 2.5 mM MBCD. Stimulation is performed after 60 s with soluble HEL. Experiment shown is representative of five independent experiments.

Techniques Used: Flow Cytometry, Cytometry, Fluorescence, Staining

72) Product Images from "Extracellular Bacterial Pathogen Induces Host Cell Surface Reorganization to Resist Shear Stress"

Article Title: Extracellular Bacterial Pathogen Induces Host Cell Surface Reorganization to Resist Shear Stress

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000314

Lipid microdomain disruption by cholesterol depletion prevents bacteria-induced cellular response. The effect of cholesterol depletion by methyl-ß-cyclodextrin (MßCD) on the interaction of N. meningitidis with host cells was tested. (A) Ability of N. meningitidis microcolonies to recruit cellular components determined by immunofluorescence using Ezrin as a marker. Bacteria and nuclei were stained with DAPI (DAPI); Ezrin was detected with anti-Ezrin polyclonal anti-serum (Ezrin); and images were merged (Merge). Scale bar corresponds to 10 µm. The top set of images are untreated cells and in the bottom set, cells were treated with MßCD. (B) Frequency of bacterial microcolonies efficiently recruiting ezrin (recruitment index) for non-treated cells (NT), in the presence of Cytochalasin D (CD), Nocodazole (Noco), and MßCD. (C) Dose response effect of MßCD with regard to the ability of bacterial microcolonies to reorganize the cellular surface on the surface of epithelial cells (full circles) and on endothelial cells (open circles). To control that the effect of MßCD was due to cholesterol, repletion experiments with added cholesterol were performed with both cell types (squares, open for endothelial cells and full for epithelial cells). (D) Cholesterol localization under bacterial microcolonies. GFP-expressing bacteria were used (Bacteria); Cholesterol was detected with Filipin (Cholesterol); and images were merged (Merge). The scale bar corresponds to 5 µm.
Figure Legend Snippet: Lipid microdomain disruption by cholesterol depletion prevents bacteria-induced cellular response. The effect of cholesterol depletion by methyl-ß-cyclodextrin (MßCD) on the interaction of N. meningitidis with host cells was tested. (A) Ability of N. meningitidis microcolonies to recruit cellular components determined by immunofluorescence using Ezrin as a marker. Bacteria and nuclei were stained with DAPI (DAPI); Ezrin was detected with anti-Ezrin polyclonal anti-serum (Ezrin); and images were merged (Merge). Scale bar corresponds to 10 µm. The top set of images are untreated cells and in the bottom set, cells were treated with MßCD. (B) Frequency of bacterial microcolonies efficiently recruiting ezrin (recruitment index) for non-treated cells (NT), in the presence of Cytochalasin D (CD), Nocodazole (Noco), and MßCD. (C) Dose response effect of MßCD with regard to the ability of bacterial microcolonies to reorganize the cellular surface on the surface of epithelial cells (full circles) and on endothelial cells (open circles). To control that the effect of MßCD was due to cholesterol, repletion experiments with added cholesterol were performed with both cell types (squares, open for endothelial cells and full for epithelial cells). (D) Cholesterol localization under bacterial microcolonies. GFP-expressing bacteria were used (Bacteria); Cholesterol was detected with Filipin (Cholesterol); and images were merged (Merge). The scale bar corresponds to 5 µm.

Techniques Used: Immunofluorescence, Marker, Staining, Expressing

73) Product Images from "Quantitative Comparison of the Efficacy of Various Compounds in Lowering Intracellular Cholesterol Levels in Niemann-Pick Type C Fibroblasts"

Article Title: Quantitative Comparison of the Efficacy of Various Compounds in Lowering Intracellular Cholesterol Levels in Niemann-Pick Type C Fibroblasts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048561

Dose Response Curves for Cholesterol-Lowering Efficacy as Demonstrated by LSO Ratio Values. NPC1 mutant fibroblasts were treated with various drugs for 48 h, after which they were fixed, filipin stained, imaged, and processed to give an LSO ratio value as described in Materials and Methods. Vorinostat, panobinostat, and β-cyclodextrin were very effective at lowering cholesterol levels, obtaining LSO ratios below 0.5 at several doses. Rapamycin was somewhat effective, consistently obtaining LSO ratios of ∼0.8. Decitabine had no significant effect on intracellular cholesterol levels. Chloroquine and chlorpromazine were not effective; they elevated cholesterol levels at all tested concentrations. The red lines indicate solvent controls (LSO ratio = 1). Each data point represents the average of at least three independent experiments totaling 36 images (three experiments×twelve images/experiment) and error bars are +/− SE. Data points without error bars reflect the results of one experiment; cells were not viable in two of three trials at chlorpromazine concentrations of 40 and 80 µM.
Figure Legend Snippet: Dose Response Curves for Cholesterol-Lowering Efficacy as Demonstrated by LSO Ratio Values. NPC1 mutant fibroblasts were treated with various drugs for 48 h, after which they were fixed, filipin stained, imaged, and processed to give an LSO ratio value as described in Materials and Methods. Vorinostat, panobinostat, and β-cyclodextrin were very effective at lowering cholesterol levels, obtaining LSO ratios below 0.5 at several doses. Rapamycin was somewhat effective, consistently obtaining LSO ratios of ∼0.8. Decitabine had no significant effect on intracellular cholesterol levels. Chloroquine and chlorpromazine were not effective; they elevated cholesterol levels at all tested concentrations. The red lines indicate solvent controls (LSO ratio = 1). Each data point represents the average of at least three independent experiments totaling 36 images (three experiments×twelve images/experiment) and error bars are +/− SE. Data points without error bars reflect the results of one experiment; cells were not viable in two of three trials at chlorpromazine concentrations of 40 and 80 µM.

Techniques Used: Mutagenesis, Staining

Dose Response Curves for Cholesterol-Lowering Efficacy of Tested Drugs in Combination with β-Cyclodextrin. NPC1 mutant fibroblasts were treated with vorinostat, panobinostat, rapamycin, and decitabine, both with and without 200 µM β-cyclodextrin, for 48 h, after which they were fixed, filipin stained, imaged, and processed to give an LSO ratio value as described in Materials and Methods. β-Cyclodextrin increased the impact vorinostat, panobinostat, rapamycin, and decitabine had on late endosomal and lysosomal cholesterol levels at all concentrations. The red lines indicate vehicle controls (LSO ratio = 1), while green lines indicate the average result of treatment with only 200 µM β-cyclodextrin (LSO ratio = 0.321). Each point represents the average of at least three independent experiments totaling 36 images (three experiments×twelve images/experiment) and error bars are +/− SE.
Figure Legend Snippet: Dose Response Curves for Cholesterol-Lowering Efficacy of Tested Drugs in Combination with β-Cyclodextrin. NPC1 mutant fibroblasts were treated with vorinostat, panobinostat, rapamycin, and decitabine, both with and without 200 µM β-cyclodextrin, for 48 h, after which they were fixed, filipin stained, imaged, and processed to give an LSO ratio value as described in Materials and Methods. β-Cyclodextrin increased the impact vorinostat, panobinostat, rapamycin, and decitabine had on late endosomal and lysosomal cholesterol levels at all concentrations. The red lines indicate vehicle controls (LSO ratio = 1), while green lines indicate the average result of treatment with only 200 µM β-cyclodextrin (LSO ratio = 0.321). Each point represents the average of at least three independent experiments totaling 36 images (three experiments×twelve images/experiment) and error bars are +/− SE.

Techniques Used: Mutagenesis, Staining

Representative Images of NPC1 Mutant Cells After Treatment with Various Compounds. GM03123 NPC1 mutant fibroblasts were treated with various drugs and combinations of drugs with 200 µM β-cyclodextrin for 48 hours, after which they were fixed, filipin stained, and imaged under UV excitation as described in Materials and Methods. Images are shown after contrast adjustment but prior to threshold application. The concentrations used in each displayed image for vorinostat, panobinostat, β-cyclodextrin, rapamycin, decitabine, chloroquine, and chlorpromazine were 10 µM, 75 nM, 200 µM, 7.5 nM, 0.625 µM, 50 µM, and 20 µM, respectively. Reduced cholesterol levels, as evidenced through decreased filipin fluorescence relative to solvent controls, are clearly observed in the vorinostat, panobinostat, β-cyclodextrin, and rapamycin treated cells. Further diminished fluorescence is observed in cells treated with both the drug and β-cyclodextrin (Drug + β-CD). No visibly obvious decrease in fluorescence is observed between cells treated with decitabine, chloroquine, or chlorpromazine, and their respective controls.
Figure Legend Snippet: Representative Images of NPC1 Mutant Cells After Treatment with Various Compounds. GM03123 NPC1 mutant fibroblasts were treated with various drugs and combinations of drugs with 200 µM β-cyclodextrin for 48 hours, after which they were fixed, filipin stained, and imaged under UV excitation as described in Materials and Methods. Images are shown after contrast adjustment but prior to threshold application. The concentrations used in each displayed image for vorinostat, panobinostat, β-cyclodextrin, rapamycin, decitabine, chloroquine, and chlorpromazine were 10 µM, 75 nM, 200 µM, 7.5 nM, 0.625 µM, 50 µM, and 20 µM, respectively. Reduced cholesterol levels, as evidenced through decreased filipin fluorescence relative to solvent controls, are clearly observed in the vorinostat, panobinostat, β-cyclodextrin, and rapamycin treated cells. Further diminished fluorescence is observed in cells treated with both the drug and β-cyclodextrin (Drug + β-CD). No visibly obvious decrease in fluorescence is observed between cells treated with decitabine, chloroquine, or chlorpromazine, and their respective controls.

Techniques Used: Mutagenesis, Staining, Fluorescence

74) Product Images from "Adenovirus RID-? activates an autonomous cholesterol regulatory mechanism that rescues defects linked to Niemann-Pick disease type C"

Article Title: Adenovirus RID-? activates an autonomous cholesterol regulatory mechanism that rescues defects linked to Niemann-Pick disease type C

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200903039

RID-α expression rescues cholesterol trafficking and associated defects in NPC cells by a class III PI3K–dependent mechanism. (a–f) NPC cells transfected in the absence or presence of PI3K inhibitors, as indicated, and stained for LAMP1, filipin, and RID-α (a, c, and e) or LAMP1 and filipin (b, d, and f). (g) Quantification of normalized filipin fluorescence intensity in cells treated similarly as in a–f and as described in Materials and methods. Data are presented as mean ± SEM (*, P
Figure Legend Snippet: RID-α expression rescues cholesterol trafficking and associated defects in NPC cells by a class III PI3K–dependent mechanism. (a–f) NPC cells transfected in the absence or presence of PI3K inhibitors, as indicated, and stained for LAMP1, filipin, and RID-α (a, c, and e) or LAMP1 and filipin (b, d, and f). (g) Quantification of normalized filipin fluorescence intensity in cells treated similarly as in a–f and as described in Materials and methods. Data are presented as mean ± SEM (*, P

Techniques Used: Expressing, Transfection, Staining, Fluorescence

RID-α (C67S) induces the formation of enlarged lipid-filled LAMP1 structures. (a) Confocal images of CHO cell lines stained with LAMP1 antibody and filipin. (b and c) Magnified images of single and merged channels from CHO–RID-α (C67S) cells stained with LBPA antibody and filipin (b) or LBPA antibody after incubation with Alexa Fluor 647 CT-B (c). (d) CHO cell lines treated with U18666A for 8 h and stained with LAMP1 antibody and filipin. (e) Cholesterol quantification in CHO cell lines treated with DMSO (vehicle) or U18666A for 8 h using the Amplex red cholesterol assay kit. Values were normalized to total cellular protein and are displayed as mean ± SEM (*, P
Figure Legend Snippet: RID-α (C67S) induces the formation of enlarged lipid-filled LAMP1 structures. (a) Confocal images of CHO cell lines stained with LAMP1 antibody and filipin. (b and c) Magnified images of single and merged channels from CHO–RID-α (C67S) cells stained with LBPA antibody and filipin (b) or LBPA antibody after incubation with Alexa Fluor 647 CT-B (c). (d) CHO cell lines treated with U18666A for 8 h and stained with LAMP1 antibody and filipin. (e) Cholesterol quantification in CHO cell lines treated with DMSO (vehicle) or U18666A for 8 h using the Amplex red cholesterol assay kit. Values were normalized to total cellular protein and are displayed as mean ± SEM (*, P

Techniques Used: Staining, Incubation, Amplex Red Cholesterol Assay

RID-α counterbalances Ad-induced autophagy. (a) Ad2 internalized by clathrin-mediated endocytosis escapes from EEs by membrane lysis followed by MT-dependent transport to the nucleus and viral gene expression. (b) RID-α, which is expressed within the first few hours of an acute infection, facilitates targeted degradation of EGFRs (blue) and proapoptotic receptors (yellow). (c) Confocal images of A549 cells infected with wild-type or RID-α–null Ad2 viruses stained with E1B and RID-α antibodies. Arrowheads indicate RID-α–positive compartments. (d) Equal aliquots of total cellular protein from mock- and Ad-infected A549 cells resolved by SDS-PAGE and immunoblotted with E1A antibody. (e–g) Confocal images of mock (e), Ad2 (f), and RID-α–null Ad2-infected (g) A549 cells stained with EEA1 and LAMP1 antibodies and filipin; magnified images of single and merged channels are shown on the right. (h) Confocal images of RID-α–null Ad2-infected A549 cells stained for LC3 and LAMP1. (e–h) Cell and nucleus (Nu) boundaries were drawn using MetaMorph software. Boxed areas show regions of the image that were magnified. (i) Equal aliquots of total cellular protein from mock- and Ad-infected cells immunoblotted with antibodies to p62/SQSTM1, EEA1, and actin as a function of time postinfection (p.i.). CCV, clathrin-coated vesicle; IB, immunoblot. (d and i) Molecular mass is indicated in kilodaltons. Bars, 10 µm.
Figure Legend Snippet: RID-α counterbalances Ad-induced autophagy. (a) Ad2 internalized by clathrin-mediated endocytosis escapes from EEs by membrane lysis followed by MT-dependent transport to the nucleus and viral gene expression. (b) RID-α, which is expressed within the first few hours of an acute infection, facilitates targeted degradation of EGFRs (blue) and proapoptotic receptors (yellow). (c) Confocal images of A549 cells infected with wild-type or RID-α–null Ad2 viruses stained with E1B and RID-α antibodies. Arrowheads indicate RID-α–positive compartments. (d) Equal aliquots of total cellular protein from mock- and Ad-infected A549 cells resolved by SDS-PAGE and immunoblotted with E1A antibody. (e–g) Confocal images of mock (e), Ad2 (f), and RID-α–null Ad2-infected (g) A549 cells stained with EEA1 and LAMP1 antibodies and filipin; magnified images of single and merged channels are shown on the right. (h) Confocal images of RID-α–null Ad2-infected A549 cells stained for LC3 and LAMP1. (e–h) Cell and nucleus (Nu) boundaries were drawn using MetaMorph software. Boxed areas show regions of the image that were magnified. (i) Equal aliquots of total cellular protein from mock- and Ad-infected cells immunoblotted with antibodies to p62/SQSTM1, EEA1, and actin as a function of time postinfection (p.i.). CCV, clathrin-coated vesicle; IB, immunoblot. (d and i) Molecular mass is indicated in kilodaltons. Bars, 10 µm.

Techniques Used: Lysis, Expressing, Infection, Staining, SDS Page, Software

75) Product Images from "Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism"

Article Title: Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism

Journal: Nature Communications

doi: 10.1038/ncomms11919

Cholesterol is redistributed to late endosomes/lysosomes in VPS35 KD HeLa cells. ( a ) Western blot analysis of VPS35 levels in HeLa cells transfected with scramble or VPS35 siRNA. Tubulin was used as an equal loading marker. ( b ) Representative confocal images of HeLa cells transfected with scramble or VPS35 siRNA, immunostained for LAMP1 (magenta) and VPS35 (green) and labelled with filipin (blue). Scale bar, 5 μm. ( c ) Normalized integrated densities of filipin staining in scramble (100±5%,±indicates s.e.m., n =29 cells, three experiments) and VPS35 siRNA cells (124±9%, n =28 cells, three experiments). * P
Figure Legend Snippet: Cholesterol is redistributed to late endosomes/lysosomes in VPS35 KD HeLa cells. ( a ) Western blot analysis of VPS35 levels in HeLa cells transfected with scramble or VPS35 siRNA. Tubulin was used as an equal loading marker. ( b ) Representative confocal images of HeLa cells transfected with scramble or VPS35 siRNA, immunostained for LAMP1 (magenta) and VPS35 (green) and labelled with filipin (blue). Scale bar, 5 μm. ( c ) Normalized integrated densities of filipin staining in scramble (100±5%,±indicates s.e.m., n =29 cells, three experiments) and VPS35 siRNA cells (124±9%, n =28 cells, three experiments). * P

Techniques Used: Western Blot, Transfection, Marker, Staining

Cholesterol is redistributed to late endosomes/lysosomes in Arf6 KO MEFs. ( a ) Western blot analysis of endogenous Arf6 levels in Arf6 Flox/Flox ; Cre-ER MEFs treated with DMSO ( Arf6 WT) or tamoxifen ( Arf6 KO). Tubulin was used as an equal loading marker. ( b ) Cholesterol levels measured by LC–MS were similar in Arf6 WT (19.1±0.7 molar % of total lipidome,±indicates s.e.m., n =11) and KO cells (18.5±0.5 molar % of total lipidome, n =12). NS denotes P > 0.05 in Student's t -test. ( c ) Representative confocal images of Arf6 WT and KO MEFs stained with filipin. Scale bar, 5 μm. ( d ) Normalized integrated densities of filipin staining were similar in Arf6 WT (100±8%,±indicates s.e.m., n=32 cells from three experiments) and Arf6 KO cells (98±8%, n =42 cells from four experiments). NS denotes P > 0.05 in Student's t -test. ( e ) Quantification of filipin puncta in Arf6 WT and KO cells. Left panel, number of puncta per cell in Arf6 WT (13±2 puncta per cell,±indicates s.e.m., n =39 cells from 3 experiments) and Arf6 KO cells (26±3 puncta per cell, n =29 cells from 3 experiments). ** P
Figure Legend Snippet: Cholesterol is redistributed to late endosomes/lysosomes in Arf6 KO MEFs. ( a ) Western blot analysis of endogenous Arf6 levels in Arf6 Flox/Flox ; Cre-ER MEFs treated with DMSO ( Arf6 WT) or tamoxifen ( Arf6 KO). Tubulin was used as an equal loading marker. ( b ) Cholesterol levels measured by LC–MS were similar in Arf6 WT (19.1±0.7 molar % of total lipidome,±indicates s.e.m., n =11) and KO cells (18.5±0.5 molar % of total lipidome, n =12). NS denotes P > 0.05 in Student's t -test. ( c ) Representative confocal images of Arf6 WT and KO MEFs stained with filipin. Scale bar, 5 μm. ( d ) Normalized integrated densities of filipin staining were similar in Arf6 WT (100±8%,±indicates s.e.m., n=32 cells from three experiments) and Arf6 KO cells (98±8%, n =42 cells from four experiments). NS denotes P > 0.05 in Student's t -test. ( e ) Quantification of filipin puncta in Arf6 WT and KO cells. Left panel, number of puncta per cell in Arf6 WT (13±2 puncta per cell,±indicates s.e.m., n =39 cells from 3 experiments) and Arf6 KO cells (26±3 puncta per cell, n =29 cells from 3 experiments). ** P

Techniques Used: Western Blot, Marker, Liquid Chromatography with Mass Spectroscopy, Staining

Lowering PI4P levels through GFP-PIPKIγi1 or GFP-Sac2 overexpression rescues cholesterol redistribution in Arf6 KO cells. ( a ) Left, representative confocal images of Arf6 KO cells expressing the K188A mutant (top) or the wild-type (bottom) GFP-PIPKIγi1 (green) labelled with filipin (blue). Scale bar, 5 μm. Center, quantification of the absolute number and relative distribution of filipin puncta in Arf6 KO cells expressing K188A GFP-PIPKIγi1 (31±4 puncta per cell,±indicates s.e.m., n =24 cells, three experiments) or WT GFP-PIPKIγi1 (18±2 puncta per cell, n =38 cells, five experiments). ** P
Figure Legend Snippet: Lowering PI4P levels through GFP-PIPKIγi1 or GFP-Sac2 overexpression rescues cholesterol redistribution in Arf6 KO cells. ( a ) Left, representative confocal images of Arf6 KO cells expressing the K188A mutant (top) or the wild-type (bottom) GFP-PIPKIγi1 (green) labelled with filipin (blue). Scale bar, 5 μm. Center, quantification of the absolute number and relative distribution of filipin puncta in Arf6 KO cells expressing K188A GFP-PIPKIγi1 (31±4 puncta per cell,±indicates s.e.m., n =24 cells, three experiments) or WT GFP-PIPKIγi1 (18±2 puncta per cell, n =38 cells, five experiments). ** P

Techniques Used: Over Expression, Expressing, Mutagenesis

Related Articles

Amplification:

Article Title: Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †
Article Snippet: Staining of lipid rafts with filipin III (Sigma) was done as described previously , as was the procedure for visualizing FM4-64 uptake ( ). .. To obtain mutant and wild-type strains expressing RVS167-GFP and GFP-GSC1 , Ura− strains were transformed with 10 μg of pU165 linearized with BglII and with an 8.4-kb fragment obtained by PCR amplification of pU191 using UO156 and UO157.

Mass Spectrometry:

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates
Article Snippet: Filipin staining and image analysis Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature. .. Images were acquired with a CoolSnap K4 CCD camera (Photometrics) via Micro-manager with the same acquisition settings (exposure time = 300 ms and gain = 1) across each experimental group.

Cytometry:

Article Title:
Article Snippet: Cellular cholesterol distribution was determined by fixing cells in 3% paraformaldehyde and then staining them in 0.05 mg/ml Filipin solution (Sigma-Aldrich). .. MM, MCL, and HeLa cells were imaged using an ImageStreamX Mark II Imaging Flow Cytometer (EMD Millipore, Danvers, MA), with 1000 cells counted per treatment, and visualized at 60× magnification.

Incubation:

Article Title: Transcriptional profile of Paracoccidioides spp. in response to itraconazole
Article Snippet: Control cells were incubated in MMcM without drug. .. A 5 mg/ml stock solution of filipin (Sigma-Aldrich) dissolved in DMSO was diluted to 25 μg/ml and used to stain the fixed samples for 10 min.

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates
Article Snippet: .. Filipin staining and image analysis Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature. .. Fluorescence imaging was performed on an Olympus IX-81 inverted microscope using a Nikon Intensilight illuminator, a Nikon Plan Apo VC 20× objective (N.A.

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: After fixation, cells were washed with PBS and incubated with 1.5 g glycine in PBS 10 min at RT. .. Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT.

Proliferation Assay:

Article Title: Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann–Pick C1 mutant cell via lysosome-associated membrane protein 1
Article Snippet: A CellTiter 96 Aqueous One Solution Cell Proliferation Assay System was purchased from Promega (Madison, WI, USA). .. Filipin III were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Cell Culture:

Article Title: Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann–Pick C1 mutant cell via lysosome-associated membrane protein 1
Article Snippet: Reagents Cell culture media and reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA); these include Dulbecco’s modified Eagle’s medium (DMEM), MEM non-essential amino acids, fetal bovine serum (FBS), penicillin, and streptomycin. .. Filipin III were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Article Title: Small GTPase Rab14 down-regulates UT-A1 urea transport activity through enhanced clathrin-dependent endocytosis
Article Snippet: Paragraph title: Cell culture, transient transfection, and treatment ... Chlorpromazine and filipin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Article Title:
Article Snippet: Brightfield images were captured in live cultured cells at 40× magnification. .. Cellular cholesterol distribution was determined by fixing cells in 3% paraformaldehyde and then staining them in 0.05 mg/ml Filipin solution (Sigma-Aldrich).

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT. .. For labeling of host cell cholesterol pool by fluorescent cholesterol species, macrophages were cultured as indicated above.

Expressing:

Article Title: Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †
Article Snippet: Staining of lipid rafts with filipin III (Sigma) was done as described previously , as was the procedure for visualizing FM4-64 uptake ( ). .. To obtain mutant and wild-type strains expressing RVS167-GFP and GFP-GSC1 , Ura− strains were transformed with 10 μg of pU165 linearized with BglII and with an 8.4-kb fragment obtained by PCR amplification of pU191 using UO156 and UO157.

Modification:

Article Title: Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann–Pick C1 mutant cell via lysosome-associated membrane protein 1
Article Snippet: Reagents Cell culture media and reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA); these include Dulbecco’s modified Eagle’s medium (DMEM), MEM non-essential amino acids, fetal bovine serum (FBS), penicillin, and streptomycin. .. Filipin III were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Western Blot:

Article Title: Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †
Article Snippet: Staining of lipid rafts with filipin III (Sigma) was done as described previously , as was the procedure for visualizing FM4-64 uptake ( ). .. Transformants were screened for the expression of Rvs167-GFP and GFP-Gsc1 by epifluorescence and/or Western blot analysis.

Transformation Assay:

Article Title: Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †
Article Snippet: Staining of lipid rafts with filipin III (Sigma) was done as described previously , as was the procedure for visualizing FM4-64 uptake ( ). .. To obtain mutant and wild-type strains expressing RVS167-GFP and GFP-GSC1 , Ura− strains were transformed with 10 μg of pU165 linearized with BglII and with an 8.4-kb fragment obtained by PCR amplification of pU191 using UO156 and UO157.

Transfection:

Article Title: Small GTPase Rab14 down-regulates UT-A1 urea transport activity through enhanced clathrin-dependent endocytosis
Article Snippet: Paragraph title: Cell culture, transient transfection, and treatment ... Chlorpromazine and filipin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Immunoprecipitation:

Article Title: Small GTPase Rab14 down-regulates UT-A1 urea transport activity through enhanced clathrin-dependent endocytosis
Article Snippet: Cells were then processed for cell surface biotinylation, immunoprecipitation or immunofluorescence microscopy. .. Chlorpromazine and filipin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Infection:

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT. .. Sixteen hours prior to infection, macrophages were incubated with 22‐NBD‐cholesterol (2.5 μmol/L, Life Technologies, Darmstadt, Germany) or TopFluor (BODIPY)‐cholesterol (0.5 μmol/L, Avanti Polar Lipids, Inc., Alabaster, USA), washed with PBS, and infected with L . mexicana amastigotes for the indicated time points.

other:

Article Title: S-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding *-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding *
Article Snippet: OA, caprylate, stearate, GSH, 1,4-dithiothreitol (DTT), and filipin III were purchased from Sigma-Aldrich.

Imaging:

Article Title:
Article Snippet: Cellular cholesterol distribution was determined by fixing cells in 3% paraformaldehyde and then staining them in 0.05 mg/ml Filipin solution (Sigma-Aldrich). .. MM, MCL, and HeLa cells were imaged using an ImageStreamX Mark II Imaging Flow Cytometer (EMD Millipore, Danvers, MA), with 1000 cells counted per treatment, and visualized at 60× magnification.

Article Title: Regulation of presynaptic strength by controlling Ca2+ channel mobility: effects of cholesterol depletion on release at the cone ribbon synapse
Article Snippet: Paragraph title: Retina tissue fixation and cholesterol imaging. ... To image membrane lipids at the terminals of cone photoreceptors, we applied filipin III (Sigma-Aldrich) to label membrane cholesterol or FITC-CTXB (Sigma-Aldrich) to label ganglioside GM1 glycoproteins associated with cholesterol-rich lipid rafts.

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates
Article Snippet: Filipin staining and image analysis Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature. .. Fluorescence imaging was performed on an Olympus IX-81 inverted microscope using a Nikon Intensilight illuminator, a Nikon Plan Apo VC 20× objective (N.A.

Inverted Microscopy:

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates
Article Snippet: Filipin staining and image analysis Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature. .. Fluorescence imaging was performed on an Olympus IX-81 inverted microscope using a Nikon Intensilight illuminator, a Nikon Plan Apo VC 20× objective (N.A.

Polymerase Chain Reaction:

Article Title: Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †
Article Snippet: Staining of lipid rafts with filipin III (Sigma) was done as described previously , as was the procedure for visualizing FM4-64 uptake ( ). .. To obtain mutant and wild-type strains expressing RVS167-GFP and GFP-GSC1 , Ura− strains were transformed with 10 μg of pU165 linearized with BglII and with an 8.4-kb fragment obtained by PCR amplification of pU191 using UO156 and UO157.

Immunofluorescence:

Article Title: Small GTPase Rab14 down-regulates UT-A1 urea transport activity through enhanced clathrin-dependent endocytosis
Article Snippet: Cells were then processed for cell surface biotinylation, immunoprecipitation or immunofluorescence microscopy. .. Chlorpromazine and filipin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Fluorescence:

Article Title: Transcriptional profile of Paracoccidioides spp. in response to itraconazole
Article Snippet: Paragraph title: Fluorescence microscopy ... A 5 mg/ml stock solution of filipin (Sigma-Aldrich) dissolved in DMSO was diluted to 25 μg/ml and used to stain the fixed samples for 10 min.

Article Title:
Article Snippet: Paragraph title: Fluorescence Microscopy and Fluorescent Staining. ... Cellular cholesterol distribution was determined by fixing cells in 3% paraformaldehyde and then staining them in 0.05 mg/ml Filipin solution (Sigma-Aldrich).

Article Title: Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †
Article Snippet: Paragraph title: Fluorescence microscopy. ... Staining of lipid rafts with filipin III (Sigma) was done as described previously , as was the procedure for visualizing FM4-64 uptake ( ).

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates
Article Snippet: Filipin staining and image analysis Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature. .. Fluorescence imaging was performed on an Olympus IX-81 inverted microscope using a Nikon Intensilight illuminator, a Nikon Plan Apo VC 20× objective (N.A.

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: Paragraph title: Fluorescence microscopy ... Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT.

Article Title: Progesterone Impairs Human Ether-a-go-go-related Gene (HERG) Trafficking by Disruption of Intracellular Cholesterol Homeostasis *
Article Snippet: .. Cells were then stained with filipin (Sigma-Aldrich) and viewed by fluorescence microscopy at magnification of ×20 or ×40 times using a UV filter set. .. An Amplex®Red Cholesterol Assay kit (Molecular Probes) was used to measure the cellular cholesterol level following the manufacturer's instructions.

Methylation:

Article Title: Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann–Pick C1 mutant cell via lysosome-associated membrane protein 1
Article Snippet: Filipin III were obtained from Sigma-Aldrich (St. Louis, MO, USA). .. These CDs include αCD, βCD, and γCD of native forms as well as their derivatives with hydroxypropyl (HP), randomly methylated (RM), and carboxymethyl (CM) substitutions.

Mutagenesis:

Article Title: Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †
Article Snippet: Staining of lipid rafts with filipin III (Sigma) was done as described previously , as was the procedure for visualizing FM4-64 uptake ( ). .. To obtain mutant and wild-type strains expressing RVS167-GFP and GFP-GSC1 , Ura− strains were transformed with 10 μg of pU165 linearized with BglII and with an 8.4-kb fragment obtained by PCR amplification of pU191 using UO156 and UO157.

CtB Assay:

Article Title: Therapeutics to Promote CNS Repair: A Natural Human Neuron-Binding IgM Regulates Membrane-Raft Dynamics and Improves Motility in a Mouse Model of Multiple Sclerosis
Article Snippet: .. Anti-β3-tubulin (Promega); EDTA, poly-D-lysine, and filipin (Sigma); Anti-Tau1 and anti-Map2 (Millipore); Texas Red phalloidin, cholera toxin B (CTB, Alexa Fluor 594), Neurobasal Medium and B27 (Invitrogen). .. SJL/J mice (Jackson Laboratories, Bar Harbor, ME) were housed and bred in Mayo Clinic’s animal care facility.

Flow Cytometry:

Article Title:
Article Snippet: Cellular cholesterol distribution was determined by fixing cells in 3% paraformaldehyde and then staining them in 0.05 mg/ml Filipin solution (Sigma-Aldrich). .. MM, MCL, and HeLa cells were imaged using an ImageStreamX Mark II Imaging Flow Cytometer (EMD Millipore, Danvers, MA), with 1000 cells counted per treatment, and visualized at 60× magnification.

Microscopy:

Article Title: Transcriptional profile of Paracoccidioides spp. in response to itraconazole
Article Snippet: Paragraph title: Fluorescence microscopy ... A 5 mg/ml stock solution of filipin (Sigma-Aldrich) dissolved in DMSO was diluted to 25 μg/ml and used to stain the fixed samples for 10 min.

Article Title: Small GTPase Rab14 down-regulates UT-A1 urea transport activity through enhanced clathrin-dependent endocytosis
Article Snippet: Cells were then processed for cell surface biotinylation, immunoprecipitation or immunofluorescence microscopy. .. Chlorpromazine and filipin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Article Title:
Article Snippet: Paragraph title: Fluorescence Microscopy and Fluorescent Staining. ... Cellular cholesterol distribution was determined by fixing cells in 3% paraformaldehyde and then staining them in 0.05 mg/ml Filipin solution (Sigma-Aldrich).

Article Title: Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †
Article Snippet: Paragraph title: Fluorescence microscopy. ... Staining of lipid rafts with filipin III (Sigma) was done as described previously , as was the procedure for visualizing FM4-64 uptake ( ).

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: Paragraph title: Fluorescence microscopy ... Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT.

Article Title: Progesterone Impairs Human Ether-a-go-go-related Gene (HERG) Trafficking by Disruption of Intracellular Cholesterol Homeostasis *
Article Snippet: .. Cells were then stained with filipin (Sigma-Aldrich) and viewed by fluorescence microscopy at magnification of ×20 or ×40 times using a UV filter set. .. An Amplex®Red Cholesterol Assay kit (Molecular Probes) was used to measure the cellular cholesterol level following the manufacturer's instructions.

Labeling:

Article Title: Histone deacetylase inhibitors modulate KATP subunit transcription in HL-1 cardiomyocytes through effects on cholesterol homeostasis
Article Snippet: .. Fixed cells were labeled with filipin (0.05 mg/mL in PBS, Sigma) and PicoGreen (1:1000, Molecular Probes) for 1 h at room temperature to label non-esterified cholesterol and DNA, respectively. ..

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT. .. For labeling of host cell cholesterol pool by fluorescent cholesterol species, macrophages were cultured as indicated above.

Plasmid Preparation:

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT. .. After staining, cells were washed three times with PBS and the cover slips were mounted in Vectashield HardSet Mounting Medium (Vector Laboratories, Burlingame, USA).

LDH Cytotoxicity Assay:

Article Title: Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann–Pick C1 mutant cell via lysosome-associated membrane protein 1
Article Snippet: An LDH Cytotoxicity Assay Kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). .. Filipin III were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Concentration Assay:

Article Title: Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †
Article Snippet: Cytochalasin A was added where indicated to a final concentration of 5 μM. .. Staining of lipid rafts with filipin III (Sigma) was done as described previously , as was the procedure for visualizing FM4-64 uptake ( ).

Staining:

Article Title: Transcriptional profile of Paracoccidioides spp. in response to itraconazole
Article Snippet: .. A 5 mg/ml stock solution of filipin (Sigma-Aldrich) dissolved in DMSO was diluted to 25 μg/ml and used to stain the fixed samples for 10 min. .. Samples were then rinsed with ddH2 O, mounted on a microscope slide and sealed with nail varnish.

Article Title:
Article Snippet: .. Cellular cholesterol distribution was determined by fixing cells in 3% paraformaldehyde and then staining them in 0.05 mg/ml Filipin solution (Sigma-Aldrich). .. Cells were then washed, mounted in ProLong anti-fade reagent (Invitrogen), and visualized by ultraviolet fluorescence, and images were captured at 40×.

Article Title: Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways †
Article Snippet: .. Staining of lipid rafts with filipin III (Sigma) was done as described previously , as was the procedure for visualizing FM4-64 uptake ( ). .. To obtain mutant and wild-type strains expressing RVS167-GFP and GFP-GSC1 , Ura− strains were transformed with 10 μg of pU165 linearized with BglII and with an 8.4-kb fragment obtained by PCR amplification of pU191 using UO156 and UO157.

Article Title: Membrane cholesterol mediates the cellular effects of monolayer graphene substrates
Article Snippet: .. Filipin staining and image analysis Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, washed, and incubated with filipin (1:500 in PBS, Sigma-Aldrich) for 2 h at room temperature. .. Fluorescence imaging was performed on an Olympus IX-81 inverted microscope using a Nikon Intensilight illuminator, a Nikon Plan Apo VC 20× objective (N.A.

Article Title: Changes to cholesterol trafficking in macrophages by Leishmania parasites infection. Changes to cholesterol trafficking in macrophages by Leishmania parasites infection
Article Snippet: .. Staining was performed with PBS/10% FCS containing 0.05 mg ml−1 filipin (25 mg ml−1 in DMSO, Sigma‐Aldrich, St. Louis, USA) for 1 hr at RT. .. After staining, cells were washed three times with PBS and the cover slips were mounted in Vectashield HardSet Mounting Medium (Vector Laboratories, Burlingame, USA).

Article Title: Progesterone Impairs Human Ether-a-go-go-related Gene (HERG) Trafficking by Disruption of Intracellular Cholesterol Homeostasis *
Article Snippet: .. Cells were then stained with filipin (Sigma-Aldrich) and viewed by fluorescence microscopy at magnification of ×20 or ×40 times using a UV filter set. .. An Amplex®Red Cholesterol Assay kit (Molecular Probes) was used to measure the cellular cholesterol level following the manufacturer's instructions.

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    Millipore filipin
    StarD5 deletion lowers PM cholesterol in macrophages and ABCA1-dependent cholesterol efflux. A: Primary peritoneal macrophages from WT and StarD5 −/− mice were imaged following staining with <t>filipin</t> and BODIPY 493-503, as indicated, as well as by differential interference contrast for those stained with BODIPY 493-503. B: Total, free, esterified, and PM cholesterol were quantified as described in the Materials and Methods (n = 4). C: Accessible PM cholesterol was quantified as described in the Materials and Methods (n = 3, * P ≤ 0.05 for WT versus StarD5 −/− ). D: Primary peritoneal macrophages from WT and StarD5 −/− mice were used in cholesterol efflux assays as described in the Materials and Methods in the absence or presence of cAMP or cyclosporine A. Cholesterol efflux was calculated as the percentage of total cell 3 H-cholesterol content, which was about 5% of the total cholesterol (total effluxed 3 H-cholesterol + cell-associated 3 H-cholesterol) and represented as a percentage of the effluxed cholesterol in WT macrophages in the presence of cAMP. Primary peritoneal macrophages from StarD5 −/− mice infected with Ad-StarD5 (MOI of 200) were also used. The values represent the average ± SD (n = 4, * P
    Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    StarD5 deletion lowers PM cholesterol in macrophages and ABCA1-dependent cholesterol efflux. A: Primary peritoneal macrophages from WT and StarD5 −/− mice were imaged following staining with filipin and BODIPY 493-503, as indicated, as well as by differential interference contrast for those stained with BODIPY 493-503. B: Total, free, esterified, and PM cholesterol were quantified as described in the Materials and Methods (n = 4). C: Accessible PM cholesterol was quantified as described in the Materials and Methods (n = 3, * P ≤ 0.05 for WT versus StarD5 −/− ). D: Primary peritoneal macrophages from WT and StarD5 −/− mice were used in cholesterol efflux assays as described in the Materials and Methods in the absence or presence of cAMP or cyclosporine A. Cholesterol efflux was calculated as the percentage of total cell 3 H-cholesterol content, which was about 5% of the total cholesterol (total effluxed 3 H-cholesterol + cell-associated 3 H-cholesterol) and represented as a percentage of the effluxed cholesterol in WT macrophages in the presence of cAMP. Primary peritoneal macrophages from StarD5 −/− mice infected with Ad-StarD5 (MOI of 200) were also used. The values represent the average ± SD (n = 4, * P

    Journal: Journal of Lipid Research

    Article Title: StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis [S]

    doi: 10.1194/jlr.M091967

    Figure Lengend Snippet: StarD5 deletion lowers PM cholesterol in macrophages and ABCA1-dependent cholesterol efflux. A: Primary peritoneal macrophages from WT and StarD5 −/− mice were imaged following staining with filipin and BODIPY 493-503, as indicated, as well as by differential interference contrast for those stained with BODIPY 493-503. B: Total, free, esterified, and PM cholesterol were quantified as described in the Materials and Methods (n = 4). C: Accessible PM cholesterol was quantified as described in the Materials and Methods (n = 3, * P ≤ 0.05 for WT versus StarD5 −/− ). D: Primary peritoneal macrophages from WT and StarD5 −/− mice were used in cholesterol efflux assays as described in the Materials and Methods in the absence or presence of cAMP or cyclosporine A. Cholesterol efflux was calculated as the percentage of total cell 3 H-cholesterol content, which was about 5% of the total cholesterol (total effluxed 3 H-cholesterol + cell-associated 3 H-cholesterol) and represented as a percentage of the effluxed cholesterol in WT macrophages in the presence of cAMP. Primary peritoneal macrophages from StarD5 −/− mice infected with Ad-StarD5 (MOI of 200) were also used. The values represent the average ± SD (n = 4, * P

    Article Snippet: Filipin was from Cayman Biochemicals; cholesterol oxidase was from Calbiochem, BODIPY 493/503 was from Fisher Scientific; cAMP, cyclosporin A, and other general chemicals were from Sigma.

    Techniques: Mouse Assay, Staining, Infection

    Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. ( A ) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL filipin III for 1 hr prior to the addition of O-1602 (10 −5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Anandamide exerts its antiproliferative actions on cholangiocarcinoma by activation of the GPR55 receptor

    doi: 10.1038/labinvest.2011.62

    Figure Lengend Snippet: Disruption of lipid rafts inhibits the antiproliferative effects of GPR55 activation. ( A ) Mz-ChA-1 cells were pretreated with lipid raft disrupters 0.1 mM β–methylcyclodextrin, or 1 μg/mL filipin III for 1 hr prior to the addition of O-1602 (10 −5 M). Cell viability was determined by MTS assays. Data are expressed as average ± SEM (*p

    Article Snippet: The inhibitors utilized were the lipid raft disruptors, methyl-β-cyclodextrin (0.1 mM), , and filipin III (1 mg/mL) ( , ), and the JNK inhibitor (10−7 M; SP600125; EMD Chemicals, Gibbstown, NJ) ( ).

    Techniques: Activation Assay

    Overall structures of CYP105P1-filipin I complex ( A ) and unliganded CYP105D6 ( B ), illustrated by ribbon representation. Heme and ligands are shown as stick models. The BC and FG loop regions are shown in dark gray. a.a. , amino acids.

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Overall structures of CYP105P1-filipin I complex ( A ) and unliganded CYP105D6 ( B ), illustrated by ribbon representation. Heme and ligands are shown as stick models. The BC and FG loop regions are shown in dark gray. a.a. , amino acids.

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques:

    Spectral changes of CYP105P1 (ferric resting state) upon the addition of increasing concentrations of filipin I ( A ), its difference spectra ( B ), and the titration curve calculated using the values of absorption differences at 387 and 422 nm ( C ).

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Spectral changes of CYP105P1 (ferric resting state) upon the addition of increasing concentrations of filipin I ( A ), its difference spectra ( B ), and the titration curve calculated using the values of absorption differences at 387 and 422 nm ( C ).

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques: Titration

    Interactions between CYP105P1 and filipin I. A , shown is an F obs − F calc omit electron density map of the filipin I molecule contoured at 4.0 σ. B , hydrophobic interactions are observed at both sides of the 28-membered ring. Labels for atoms of filipin I are underlined. C , a stereographic figure shows interactions with the BC loop, FG helices, and I helix. The water molecules mediate hydrophilic interactions with the polyol group of filipin I. The extensive hydrogen-bonding network and residues involved in it are shown as dotted lines and stick models, respectively. The distance between the C26 atom of filipin I and the heme iron is 5.0 Å, and the pro-S hydrogen side is directed toward the heme.

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Interactions between CYP105P1 and filipin I. A , shown is an F obs − F calc omit electron density map of the filipin I molecule contoured at 4.0 σ. B , hydrophobic interactions are observed at both sides of the 28-membered ring. Labels for atoms of filipin I are underlined. C , a stereographic figure shows interactions with the BC loop, FG helices, and I helix. The water molecules mediate hydrophilic interactions with the polyol group of filipin I. The extensive hydrogen-bonding network and residues involved in it are shown as dotted lines and stick models, respectively. The distance between the C26 atom of filipin I and the heme iron is 5.0 Å, and the pro-S hydrogen side is directed toward the heme.

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques:

    Active site structures of CYP105P1-filipin I ( green ) superimposed with CYP105D6 ( A , cyan ), P450cam-camphor-O 2 ( B , yellow ), and P450 EryK-erythromycin D ( C , magenta ). Water molecules and the hydroxylation target positions of substrates (C26 of filipin I, C5 of camphor, and C12 of erythromycin D) are shown as spheres . A water molecule is positioned 2.8 Å from the heme iron in the CYP105D6 structure ( A ).

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Active site structures of CYP105P1-filipin I ( green ) superimposed with CYP105D6 ( A , cyan ), P450cam-camphor-O 2 ( B , yellow ), and P450 EryK-erythromycin D ( C , magenta ). Water molecules and the hydroxylation target positions of substrates (C26 of filipin I, C5 of camphor, and C12 of erythromycin D) are shown as spheres . A water molecule is positioned 2.8 Å from the heme iron in the CYP105D6 structure ( A ).

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques:

    Closing motion of CYP105P1. Shown is a stereographic superimposition of the filipin I complex structure with the ligand-free wild-type ( A ) and H72A mutant ( B ) structures. BC loop and FG helices are colored magenta and green in ligand-free and complex structures, respectively. The filipin I molecule is shown as yellow sticks . In the ligand-free wild-type structure ( A ), the side chain of His-72 is ligated to the heme iron as the sixth ligand, and the BC loop sinks into the heme to completely cover the distal pocket.

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Closing motion of CYP105P1. Shown is a stereographic superimposition of the filipin I complex structure with the ligand-free wild-type ( A ) and H72A mutant ( B ) structures. BC loop and FG helices are colored magenta and green in ligand-free and complex structures, respectively. The filipin I molecule is shown as yellow sticks . In the ligand-free wild-type structure ( A ), the side chain of His-72 is ligated to the heme iron as the sixth ligand, and the BC loop sinks into the heme to completely cover the distal pocket.

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques: Mutagenesis

    Spectral changes of CYP105D6 (ferric resting state) upon the addition of increasing concentrations of filipin I ( A ), its difference spectra ( B ), and the titration curve calculated using the values of absorption differences at 387 and 420 nm ( C ). A nonlinear fitting with a quadratic equation was applied to the titration curve.

    Journal: The Journal of Biological Chemistry

    Article Title: Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis *

    doi: 10.1074/jbc.M109.092460

    Figure Lengend Snippet: Spectral changes of CYP105D6 (ferric resting state) upon the addition of increasing concentrations of filipin I ( A ), its difference spectra ( B ), and the titration curve calculated using the values of absorption differences at 387 and 420 nm ( C ). A nonlinear fitting with a quadratic equation was applied to the titration curve.

    Article Snippet: Before crystallization, filipin I was dissolved in dimethyl sulfoxide, mixed with a CYP105P1 sample, and concentrated using an Ultrafree Centrifugal Filter Device (Millipore, Billerica, MA).

    Techniques: Titration

    Internalization of the hMCRs in HEK293 cells. Cells were incubated with [ 125 I]-NDP- α -MSH and caveolae inhibitors, filipin (▼) and nystatin (▼) compared with control (|) at 37 °C for the indicated times. Acid-resistant and acid-sensitive binding were determined. Percentage internalization (acid-resistant binding) was expressed by measuring total binding at each time-point.

    Journal: Chemical biology & drug design

    Article Title: Cell Signaling and Trafficking of Human Melanocortin Receptors in Real Time Using Two-photon Fluorescence and Confocal Laser Microscopy: Differentiation of Agonists and Antagonists

    doi: 10.1111/j.1747-0285.2006.00432.x

    Figure Lengend Snippet: Internalization of the hMCRs in HEK293 cells. Cells were incubated with [ 125 I]-NDP- α -MSH and caveolae inhibitors, filipin (▼) and nystatin (▼) compared with control (|) at 37 °C for the indicated times. Acid-resistant and acid-sensitive binding were determined. Percentage internalization (acid-resistant binding) was expressed by measuring total binding at each time-point.

    Article Snippet: H-89 and Filipin were from Calbiochem (San Diego, CA, USA).

    Techniques: Incubation, Binding Assay